Lipid composition for the delivery of therapeutic cargos

ABSTRACT

A construct includes a core comprising an external surface and a plurality of pores, a cargo disposed in a pore of the plurality of pores, the cargo comprising a CRISPR Cas9 component or a nucleic acid sequence encoding a CRISPR Cas9 component; and a coating coupled to the core, wherein the coating comprises a cationic lipid, a pegylated lipid, a zwitterionic lipid, and a sterol. The coating may comprise a molar ratio of about 1 cationic lipid to 1 zwitterionic lipid to 0.9 sterol to 0.15 PEGylated lipid, wherein each molar ratio optionally varies by about plus or minus 10%. A method of treatment is also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.16/839,194, filed on Apr. 3, 2020. That prior application is hereinincorporated by reference in its entirety.

STATEMENT OF GOVERNMENT INTEREST

This invention was made with Government support under Contract No.DE-NA0003525 awarded by the United States Department of Energy/NationalNuclear Security Administration. The Government has certain rights inthe invention.

REFERENCE TO A SEQUENCE LISTING APPENDIX

A sequence listing appendix including an XML formatted file accompaniesthis application. The appendix includes a file named “150131-1.xml,”created on Feb. 3, 2023 (size of 231 kilobytes), which is herebyincorporated by reference in its entirety.

FIELD

This disclosure relates to a blend of coating materials for ananoparticle with a gene-editing agent payload.

BACKGROUND

CRISPR (clustered, regularly interspaced, short 37 palindromicrepeats)/Cas9 is a bacterial derived gene editing system that has beenrepurposed to edit specifically targeted sequences opening a new avenuewith enormous unrealized implications in health, disease prevention,diagnosis, and treatment. Efficient intracellular delivery ofCRISPR-Cas9 remains a hurdle in the advancement of this technology.Porous nanoparticles are an attractive delivery vehicle for a variety ofcargos including small molecule drugs and proteins due to the lowtoxicity, high biocompatibility, facile synthesis and amenability tochemical functionalization.

However, efficacious delivery of therapeutic agents such as CRISPR-Cas9still remains a challenge for certain classes of agents. For instance,due to delivery efficiency, certain coatings and gene-editing agentsdisplay effectiveness in cellular assays but show reduced efficacy invivo. In addition, different coatings will interact differently withdifferent types of particles and gene-editing agent payloads. Thus,there is a need for additional delivery constructs that can beconfigured to accommodate such agents.

SUMMARY

The present disclosure relates, in part, to a biocompatible, non-toxicconstruct including a core, a cargo, and a coating. The presentdisclosure demonstrates a tailored coating layer resulting in andimproved and efficient delivery of CRISPR/Cas9 using a lipid coatedmesoporous silica nanoparticle resulting in editing in over 30% oftarget cells in vitro.

In some embodiments, the core is a mesoporous nanoparticle (e.g., amesoporous silica nanoparticle). In particular embodiments, the core hasa dimension (e.g., a diameter, a width, or a length, or an effectiveaverage particle size) greater than about 50 nm (e.g., from about 50 nmto 300 nm, 50 nm to 100 nm, 50 nm to 150 nm, 50 nm to 200 nm, 50 nm to250 nm, 75 nm to 100 nm, 75 nm to 150 nm, 75 nm to 200 nm, 75 nm to 250nm, 75 nm to 300 nm, 100 nm to 150 nm, 100 nm to 200 nm, 100 nm to 250nm, 100 nm to 300 nm, 125 nm to 150 nm, 125 nm to 200 nm, 125 nm to 250nm, 125 nm to 300 nm, 150 nm to 200 nm, 150 nm to 250 nm, 150 nm to 300nm, 175 nm to 200 nm, 175 nm to 250 nm, 175 nm to 300 nm, 200 nm to 250nm, 200 nm to 300 nm, 225 nm to 250 nm, 225 nm to 300 nm, 250 nm to 300nm, or 275 nm to 300 nm).

In particular embodiments, the core includes a plurality of pores, inwhich an average dimension of the pores is sufficiently large enough toaccommodate a large cargo. In some embodiments, the average dimension isgreater than about 2 nm, (e.g., of from about 5 nm to 35 nm, includingfrom 5 nm to 10 nm, 5 nm to 15 nm, 5 nm to 20 nm, 5 nm to 25 nm, 5 nm to30 nm, 8 nm to 10 nm, 8 nm to 15 nm, 8 nm to 20 nm, 8 nm to 25 nm, 8 nmto 30 nm, 8 nm to 35 nm, 10 nm to 15 nm, 10 nm to 20 nm, 10 nm to 25 nm,10 nm to 30 nm, 10 nm to 35 nm, 12 nm to 15 nm, 12 nm to 20 nm, 12 nm to25 nm, 12 nm to 30 nm, 12 nm to 35 nm, 15 nm to 20 nm, 15 nm to 25 nm,15 nm to 30 nm, 15 nm to 35 nm, 18 nm to 20 nm, 18 nm to 25 nm, 18 nm to30 nm, 18 nm to 35 nm, 20 nm to 25 nm, 20 nm to 30 nm, 20 nm to 35 nm,25 nm to 30 nm, 25 nm to 35 nm, or 30 nm to 35 nm).

The cargo is, for example, an mRNA or a CRISPR component, such as anydescribed herein. Exemplary CRISPR components include a Cas protein, aguide nucleic acid, a plasmid, as well as combinations thereof (e.g., aribonucleoprotein complex). In some embodiments, an average dimension ofthe pores is greater than a dimension of the cargo. In otherembodiments, an average dimension of the pores is smaller than adimension of the cargo.

The construct further includes an outer layer or coating. An exemplarycoating includes a cationic lipid, a zwitterionic lipid, a PEGylatedlipid, and a sterol.

In a second aspect, a formulation including a plurality of constructs(e.g., any described herein) is disclosed along with a pharmaceuticallyacceptable excipient.

In a third aspect, a construct includes: a core comprising an externalsurface and a plurality of pores, wherein an average dimension of theplurality of pores is greater than about 2 nm. It also includes a cargodisposed in a pore of the plurality of pores, the cargo comprising oneor more selected from the group consisting of: peptides, proteins,nucleic acids, mRNA, aptamers, antibodies, pharmaceuticals,carbohydrates, dyes, and markers. It also includes a coating coupled tothe core, wherein the coating comprises a cationic lipid, a pegylatedlipid, a zwitterionic lipid, and a sterol. The coating comprises a molarratio of about 1 cationic lipid to 1 zwitterionic lipid to 0.9 sterol to0.15 PEGylated lipid, wherein each molar ratio optionally varies byabout plus or minus 10%. The the cationic lipid is1,2-dioleoyl-3-trimethylammonium-propane, the zwitterionic lipid is1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, the sterol ischolesterol, and the PEGylated lipid is1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy-(polyethyleneglycol)-2000].

In a fourth aspect, the present disclosure features a method of treatinga subject, the method including: administering to a subject in needthereof, an effective amount of a construct, the construct including: acore comprising an external surface and a plurality of pores, wherein anaverage dimension of the plurality of pores is greater than about 2 nm;a cargo disposed in a pore of the plurality of pores, the cargocomprising a CRISPR Cas9 component or a nucleic acid sequence encoding aCRISPR Cas9 component; and a coating coupled to the core. The coatingcomprises a cationic lipid, a pegylated lipid, a zwitterionic lipid, anda sterol.

In a fifth aspect, the disclosure features a method of forming theconstruct.

Definitions

As used herein, the term “about” means+/−10% of any recited value. Asused herein, this term modifies any recited value, range of values, orendpoints of one or more ranges.

By “micro” is meant having at least one dimension that is less than 1 mmbut equal to or larger than 1 μm. For instance, a microstructure (e.g.,any structure described herein, such as a microparticle) can have alength, width, height, cross-sectional dimension, circumference, radius(e.g., external or internal radius), or diameter that is less than 1 mmbut equal to or larger than 1 μm. In another instance, themicrostructure has a dimension that is of from about 1 μm to 1 mm.

By “nano” is meant having at least one dimension that is less than 1 mbut equal to or larger than 1 nm. For instance, a nanostructure (e.g.,any structure described herein, such as a nanoparticle) can have alength, width, height, cross-sectional dimension, circumference, radius(e.g., external or internal radius), or diameter that is less than 1 mbut equal to or larger than 1 nm. In another instance, the nanostructurehas a dimension that is of from about 1 nm to about 1 μm.

The term “cargo” is used herein to describe a molecule or compound,whether a small molecule or macromolecule having an activity relevant toits use in particles (e.g., a construct, a nanoparticle, or a mesoporoussilica nanoparticle), especially including biological activity, that canbe included in or with particles according to the present disclosure. Inprincipal embodiments of the present disclosure, the cargo is a nucleicacid sequence, such as double stranded (ds) plasmid DNA. The cargo maybe included within the pores, associated with the pore (e.g., by way ofa spacer), and/or on the surface of the core (e.g., by way of a spacer)according to the present disclosure. Additional representative cargo mayinclude, for example, a small molecule bioactive agent, a nucleic acid(e.g., RNA or DNA), a polypeptide, including a protein or acarbohydrate. Particular examples of such cargo include RNA, such asmRNA, siRNA, shRNA micro RNA, a polypeptide or protein, including aprotein toxin (e.g., ricin toxin A-chain or diphtheria toxin A-chain),and/or DNA (including double stranded or linear DNA, complementary DNA(cDNA), minicircle DNA, naked DNA and plasmid DNA, which optionally maybe supercoiled and/or packaged (e.g., with histones) and which may beoptionally modified with a nuclear localization sequence). Cargo mayalso include a reporter as described herein.

The phrase “effective average particle size” as used herein to describea multiparticulate (e.g., a porous nanoparticulate) means that at least50% of the particles therein are of a specified size. Accordingly,“effective average particle size of less than about 2,000 nm indiameter” means that at least 50% of the particles therein are less thanabout 2,000 nm in diameter. In certain embodiments, nanoparticulateshave an effective average particle size of less than about 2,000 nm(i.e., 2 microns), less than about 1,900 nm, less than about 1,800 nm,less than about 1,700 nm, less than about 1,600 nm, less than about1,500 nm, less than about 1,400 nm, less than about 1,300 nm, less thanabout 1,200 nm, less than about 1,100 nm, less than about 1,000 nm, lessthan about 900 nm, less than about 800 nm, less than about 700 nm, lessthan about 600 nm, less than about 500 nm, less than about 400 nm, lessthan about 300 nm, less than about 250 nm, less than about 200 nm, lessthan about 150 nm, less than about 100 nm, less than about 75 nm, orless than about 50 nm, as measured by light-scattering methods. Incertain aspects of the present disclosure, the particles aremonodisperse and generally no greater than about 50 nm in averagediameter, often less than about 30 nm in average diameter, as otherwisedescribed herein. The term “D₅₀” refers to the particle size below which50% of the particles in a multiparticulate fall. Similarly, the term“D₉₀” refers to the particle size below which 90% of the particles in amultiparticulate fall.

The term “monodisperse” is used as a standard definition established bythe National Institute of Standards and Technology (NIST) (Particle SizeCharacterization, Special Publication 960-1, January 2001) to describe adistribution of particle size (diameter) within a population ofparticles, in this case nanoparticles, which particle distribution maybe considered monodisperse if at least 90% of the distribution lieswithin 5% of the median size, measured by dynamic light scattering (DLS)that calculates particles hydrodynamic diameter and polydispersity index(PDI) using a Malvern Zetasizer. See, e.g., LaBauve, et al, Lipid-CoatedMesoporous Silica Nanoparticles for the Delivery of the ML336Antiviralto Inhibit Encephalitic Alphavirus Infection, Sci Rep. 2018; 8: 13990,2018 Sep. 18. doi: 10.1038/s41598-018-32033-w, incorporated herein byreference for more information on measurement technique.

The term “lipid” is used to describe the components which are used toform lipid mono-, bi-, or multilayers on the surface of the particles(e.g., a core of the particle), that are used in the present disclosure(e.g., as constructs) and may include a PEGylated lipid. Variousembodiments provide nanostructures, that are constructed fromnanoparticles, which support one or more lipid layers (e.g., bilayer(s)or multilayer(s)).

The terms “targeting ligand” and “targeting active species” are used todescribe a compound or moiety (e.g., an antigen), which is complexed orcovalently bonded to the surface of a particle (e.g., either directly onan outer surface of a delivery platform or on an outer layer). Thetargeting ligand, in turn, binds to a moiety on the surface of a cell tobe targeted so that the constructs may bind to the surface of thetargeted cell, enter the cell or an organelle thereof, and/or deposittheir contents into the cell. The targeting active species for use inthe present disclosure may be a targeting peptide (e.g., a receptorligand, a cell penetration peptide, a fusogenic peptide, or anendosomolytic peptide, as otherwise described herein), a polypeptideincluding an antibody or antibody fragment, an aptamer, or acarbohydrate, among other species that bind (e.g., selectively bind) toa targeted cell.

The term “reporter” is used to describe an imaging agent or moiety thatis incorporated into the outer layer or cargo of particles according toan embodiment of the present disclosure and provides a signal that canbe measured. The moiety may provide a fluorescent signal or may be aradioisotope which allows radiation detection, among others. Exemplaryfluorescent labels for use in particles (e.g., via conjugation oradsorption to the outer layer or the core, via integration into thematrix of the core, and/or via incorporation into cargo elements such asDNA, RNA-sn-glycero-3-phosphoethanolamine (Texas Red DHPE, 583/608),Alexa Fluor® 647 hydrazide (649/666), Alexa Fluor® 647 carboxylic acid,succinimidyl ester (650/668), Ulysis™ Alexa Fluor® 647 Nucleic AcidLabeling Kit (650/670), Alexa Fluor® 647 conjugate of annexin V(650/665), other fluorescent labels, colorimetric labels, quantum dots,nanoparticles, microparticles, barcodes, radio labels (e.g., RF labelsor barcodes), avidin, biotin, tags, dyes, an enzyme that can optionallyinclude one or more linking agents and/or one or more dyes, as well ascombinations thereof etc. Additional reporters can include a detectionagent (e.g., a dye, such as an electroactive detection agent, afluorescent dye, a luminescent dye, a chemiluminescent dye, acolorimetric dye, a radioactive agent, a contrast agent, etc.), aparticle (e.g., such as a microparticle, a nanoparticle, a latex bead, acolloidal particle, a magnetic particle, a fluorescent particle, etc.),and/or a label (e.g., an electroactive label, an electrocatalytic label,a fluorescent label, a colorimetric label, a quantum dot, ananoparticle, a microparticle, a barcode, a radio label (e.g., an RFlabel or barcode), avidin, biotin, a tag, a dye, a marker, an enzymethat can optionally include one or more linking agents and/or one ormore dyes). Moieties that enhance the fluorescent signal or slow thefluorescent fading may also be incorporated and include SlowFade® Goldantifade reagent (with and without DAPI) and Image-iT® FX signalenhancer. All of these are well known in the art.

The terms “polynucleotide” and “nucleic acid,” used interchangeablyherein, refer to a polymeric form of nucleotides of any length, eitherribonucleotides or deoxyribonucleotides. Thus, this term includes, butis not limited to, single-stranded (e.g., sense or antisense),double-stranded, or multi-stranded ribonucleic acids (RNAs),deoxyribonucleic acids (DNAs), threose nucleic acids (TNAs), glycolnucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids(LNAs), or hybrids thereof, genomic DNA, cDNA, DNA-RNA hybrids, or apolymer comprising purine and pyrimidine bases or other natural,chemically or biochemically modified, non-natural, or derivatizednucleotide bases. Polynucleotides can have any useful two-dimensional orthree-dimensional structure or motif, such as regions including one ormore duplex, triplex, quadruplex, hairpin, and/or pseudoknot structuresor motifs. In any nucleic acid described herein, U may be replaced withT and vice versa.

The term “modified,” as used in reference to nucleic acids, means anucleic acid sequence including one or more modifications to thenucleobase, nucleoside, nucleotide, phosphate group, sugar group, and/orinternucleoside linkage (e.g., phosphodiester backbone, linkingphosphate, or a phosphodiester linkage).

The nucleoside modification may include, but is not limited to,pyridin-4-one ribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine,2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine,5-hydroxyuridine, 3-methyluridine, 5-carboxymethyl-uridine,1-carboxymethyl-pseudouridine, 5-propynyl-uridine,1-propynyl-pseudouridine, 5-taurinomethyluridine,1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine,1-taurinomethyl-4-thio-uridine, 5-methyl-uridine,1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine,2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine,2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine,dihydropseudouridine, 2-thio-dihydrouridine,2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine,4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, 5-aza-cytidine,pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine,5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine,1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine,2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine,4-thio-1-methyl-pseudoisocytidine,4-thio-1-methyl-1-deaza-pseudoisocytidine,1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine,5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine,2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine,4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine,2-aminopurine, 2,6-diaminopurine, 7-deaza-adenine,7-deaza-8-aza-adenine, 7-deaza-2-aminopurine,7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine,7-deaza-8-aza-2,6-diaminopurine, 1-methyladenosine, N6-methyladenosine,N6-isopentenyladenosine, N6-(cis-hydroxyisopentenyl)adenosine,2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine,N6-glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine,2-methylthio-N6-threonyl carbamoyladenosine, N6,N6-dimethyladenosine,7-methyladenine, 2-methylthio-adenine, and 2-methoxy-adenine, inosine,1-methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine,7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine,6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine,6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine,1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine,8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine,N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine, andcombinations thereof.

A sugar modification may include, but is not limited to, a lockednucleic acid (LNA, in which the 2′-hydroxyl is connected by a C₁₋₆alkylene or C₁₋₆ heteroalkylene bridge to the 4′-carbon of the sameribose sugar), replacement of the oxygen in ribose (e.g., with S, Se, oralkylene, such as methylene or ethylene), addition of a double bond(e.g., to replace ribose with cyclopentenyl or cyclohexenyl), ringcontraction of ribose (e.g., to form a 4-membered ring of cyclobutane oroxetane), ring expansion of ribose (e.g., to form a 6- or 7-memberedring having an additional carbon or heteroatom, such as foranhydrohexitol, altritol, mannitol, cyclohexanyl, cyclohexenyl, andmorpholino that also has a phosphoramidate backbone), multicyclic forms(e.g., tricyclic), and “unlocked” forms, such as glycol nucleic acid(GNA) (e.g., R-GNA or S-GNA, where ribose is replaced by glycol unitsattached to phosphodiester bonds), threose nucleic acid (TNA, whereribose is replace with a-L-threofuranosyl-(3′→2′)), and peptide nucleicacid (PNA, where 2-amino-ethyl-glycine linkages replace the ribose andphosphodiester backbone). The sugar group can also contain one or morecarbons that possess the opposite stereochemical configuration than thatof the corresponding carbon in ribose. Thus, a polynucleotide moleculecan include nucleotides containing, e.g., arabinose, as the sugar.

A backbone modification may include, but is not limited to, 2′-deoxy- or2′-O-methyl modifications. A phosphate group modification may include,but is not limited to, phosphorothioate, phosphoroselenates,boranophosphates, boranophosphate esters, hydrogen phosphonates,phosphoramidates, phosphorodiamidates, alkyl or aryl phosphonates,phosphotriesters, phosphorodithioates, bridged phosphoramidates, bridgedphosphorothioates, or bridged methylene-phosphonates.

“Complementarity” or “complementary” refers to the ability of a nucleicacid to form hydrogen bond(s) with another nucleic acid sequence byeither traditional Watson-Crick or other non-traditional types, e.g.,form Watson-Crick base pairs and/or G/U base pairs, “anneal”, or“hybridize,” to another nucleic acid in a sequence-specific,antiparallel, manner (i.e., a nucleic acid specifically binds to acomplementary nucleic acid) under the appropriate in vitro and/or invivo conditions of temperature and solution ionic strength. As is knownin the art, standard Watson-Crick base-pairing includes: adenine (A)pairing with thymidine (T), adenine (A) pairing with uracil (U), andguanine (G) pairing with cytosine (C). In addition, it is also known inthe art that for hybridization between two RNA molecules (e.g., dsRNA),guanine (G) base pairs with uracil (U). A percent complementarityindicates the percentage of residues in a nucleic acid molecule whichcan form hydrogen bonds (e.g., Watson-Crick base pairing) with a secondnucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 being 50%, 60%,70%, 80%, 90%, and 100% complementary). “Perfectly complementary” meansthat all the contiguous residues of a nucleic acid sequence willhydrogen bond with the same number of contiguous residues in a secondnucleic acid sequence. “Substantially complementary” or “sufficientcomplementarity” as used herein refers to a degree of complementaritythat is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%. 97%, 98%, 99%,or 100% over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, or more nucleotides, orrefers to two nucleic acids that hybridize under stringent conditions.

As used herein, “stringent conditions” for hybridization refer toconditions under which a nucleic acid having complementarity to a targetsequence predominantly hybridizes with the target sequence, andsubstantially does not hybridize to non-target sequences. Stringentconditions are generally sequence-dependent, and vary depending on anumber of factors. In general, the longer the sequence, the higher thetemperature at which the sequence specifically hybridizes to its targetsequence. Non-limiting examples of stringent conditions are described indetail in Tijssen (1993), Laboratory Techniques In Biochemistry AndMolecular Biology-Hybridization With Nucleic Acid Probes Part 1, SecondChapter “Overview of principles of hybridization and the strategy ofnucleic acid probe assay”, Elsevier, N.Y.

“Hybridization” refers to a reaction in which one or morepolynucleotides react to form a complex that is stabilized via hydrogenbonding between the bases of the nucleotide residues. The hydrogenbonding may occur by Watson Crick base pairing, Hoogstein binding, or inany other sequence specific manner. The complex may comprise two strandsforming a duplex structure, three or more strands forming a multistranded complex, a single self-hybridizing strand, or any combinationof these. A hybridization reaction may constitute a step in a moreextensive process, such as the initiation of PCR, or the cleavage of apolynucleotide by an enzyme. A sequence capable of hybridizing with agiven sequence is referred to as the “complement” of the given sequence.

Hybridization and washing conditions are well known and exemplified inSambrook J, Fritsch E F, and Maniatis T, “Molecular Cloning: ALaboratory Manual,” Second Edition, Cold Spring Harbor Laboratory Press,Cold Spring Harbor (1989), particularly Chapter 11 and Table 11.1therein; and Sambrook J and Russell W, “Molecular Cloning: A LaboratoryManual,” Third Edition, Cold Spring Harbor Laboratory Press, Cold SpringHarbor (2001). The conditions of temperature and ionic strengthdetermine the “stringency” of the hybridization.

Hybridization requires that the two nucleic acids contain complementarysequences, although mismatches between bases are possible. Theconditions appropriate for hybridization between two nucleic acidsdepend on the length of the nucleic acids and the degree ofcomplementation, variables well known in the art. The greater the degreeof complementation between two nucleotide sequences, the greater thevalue of the melting temperature (Tm) for hybrids of nucleic acidshaving those sequences. For hybridizations between nucleic acids withshort stretches of complementarity (e.g., complementarity over 35 orless, 30 or less, 25 or less, 22 or less, 20 or less, or 18 or lessnucleotides) the position of mismatches becomes important (see Sambrooket al., supra, 11.7-11.8). Typically, the length for a hybridizablenucleic acid is at least about 10 nucleotides. Illustrative minimumlengths for a hybridizable nucleic acid are: at least about 15nucleotides; at least about 20 nucleotides; at least about 22nucleotides; at least about 25 nucleotides; and at least about 30nucleotides). Furthermore, the skilled artisan will recognize that thetemperature and wash solution salt concentration may be adjusted asnecessary, according to factors such as length of the region ofcomplementation and the degree of complementation.

It is understood in the art that the sequence of polynucleotide need notbe 100% complementary to that of its target nucleic acid to bespecifically hybridizable or hybridizable. Moreover, a polynucleotidemay hybridize over one or more segments such that intervening oradjacent segments are not involved in the hybridization event (e.g., aloop structure or hairpin structure). A polynucleotide can comprise atleast 70%, at least 80%, at least 90%, at least 95%, at least 99%, or100% sequence complementarity to a target region within the targetnucleic acid sequence to which they are targeted. For example, anantisense nucleic acid in which 18 of 20 nucleotides of the antisensecompound are complementary to a target region, and would thereforespecifically hybridize, would represent 90 percent complementarity. Inthis example, the remaining noncomplementary nucleotides may beclustered or interspersed with complementary nucleotides and need not becontiguous to each other or to complementary nucleotides. Percentcomplementarity between particular stretches of nucleic acid sequenceswithin nucleic acids can be determined routinely using BLAST programs(basic local alignment search tools) and PowerBLAST programs known inthe art (Altschul S F et al., J. Mol. Biol. 1990; 215:403-10; Zhang J etal., Genome Res. 1997; 7:649-56) or by using the Gap program (WisconsinSequence Analysis Package, Version 8 for Unix, Genetics Computer Group,University Research Park, Madison Wis.), using default settings, whichuses the algorithm of Smith T F et al., Adv. Appl. Math. 1981;2(4):482-9).

By “protein,” “peptide,” or “polypeptide,” as used interchangeably, ismeant any chain of more than two amino acids, regardless ofpost-translational modification (e.g., glycosylation orphosphorylation), constituting all or part of a naturally occurringpolypeptide or peptide, or constituting a non-naturally occurringpolypeptide or peptide, which can include coded amino acids, non-codedamino acids, modified amino acids (e.g., chemically and/or biologicallymodified amino acids), and/or modified backbones.

The term “fragment” is meant a portion of a nucleic acid or apolypeptide that is at least one nucleotide or one amino acid shorterthan the reference sequence. This portion contains, at least about 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of thereference nucleic acid molecule or polypeptide. A fragment may contain10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600,700, 800, 900, 1000, 1250, 1500, 1750, 1800 or more nucleotides; or 10,20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450,500, 550, 600, 640 amino acids or more. In another example, anypolypeptide fragment can include a stretch of at least about 5 (e.g.,about 10, about 20, about 30, about 40, about 50, or about 100) aminoacids that are at least about 40% (e.g., about 50%, about 60%, about70%, about 80%, about 90%, about 95%, about 87%, about 98%, about 99%,or about 100%) identical to any of the sequences described herein can beutilized in accordance with this disclosure. In certain embodiments, apolypeptide to be utilized in accordance with the disclosed technologyincludes 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations (e.g., one ormore conservative amino acid substitutions, as described herein). In yetanother example, any nucleic acid fragment can include a stretch of atleast about 5 (e.g., about 7, about 8, about 10, about 12, about 14,about 18, about 20, about 24, about 28, about 30, or more) nucleotidesthat are at least about 40% (about 50%, about 60%, about 70%, about 80%,about 90%, about 95%, about 87%, about 98%, about 99%, or about 100%)identical to any of the sequences described herein can be utilized inaccordance with the disclosed technology.

The term “conservative amino acid substitution” refers to theinterchangeability in proteins of amino acid residues having similarside chains (e.g., of similar size, charge, and/or polarity). Forexample, a group of amino acids having aliphatic side chains consists ofglycine, alanine, valine, leucine, and isoleucine; a group of aminoacids having aliphatic-hydroxyl side chains consists of serine andthreonine; a group of amino acids having amide containing side chainsconsisting of asparagine and glutamine; a group of amino acids havingaromatic side chains consists of phenylalanine, tyrosine, andtryptophan; a group of amino acids having basic side chains consists oflysine, arginine, and histidine; a group of amino acids having acidicside chains consists of glutamic acid and aspartic acid; and a group ofamino acids having sulfur containing side chains consists of cysteineand methionine. Exemplary conservative amino acid substitution groupsare valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine,alanine-valine, glycine-serine, glutamate-aspartate, andasparagine-glutamine.

As used herein, when a polypeptide or nucleic acid sequence is referredto as having “at least X % sequence identity” to a reference sequence,it is meant that at least X percent of the amino acids or nucleotides inthe polypeptide or nucleic acid are identical to those of the referencesequence when the sequences are optimally aligned. An optimal alignmentof sequences can be determined in various ways that are within the skillin the art, for instance, the Smith Waterman alignment algorithm (SmithT F et al., J. Mol. Biol. 1981; 147:195-7) and BLAST (Basic LocalAlignment Search Tool; Altschul S F et al., J. Mol. Biol. 1990;215:403-10). These and other alignment algorithms are accessible usingpublicly available computer software such as “Best Fit” (Smith T F etal., Adv. Appl. Math. 1981; 2(4):482-9) as incorporated into GeneMatcherPlus™ (Schwarz and Dayhof, “Atlas of Protein Sequence and Structure,”ed. Dayhoff, M. O., pp. 353-358, 1979), BLAST, BLAST-2, BLAST-P,BLAST-N, BLAST-X, WU-BLAST-2, ALIGN, ALIGN-2, CLUSTAL, T-COFFEE, MUSCLE,MAFFT, or Megalign (DNASTAR). In addition, those skilled in the art candetermine appropriate parameters for measuring alignment, including anyalgorithms needed to achieve optimal alignment over the length of thesequences being compared. In general, for polypeptides, the length ofcomparison sequences can be at least five amino acids, such as, forexample, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200,250, 300, 400, 500, 600, 700, or more amino acids, up to the entirelength of the polypeptide. For nucleic acids, the length of comparisonsequences can generally be at least 10, 20, 30, 40, 50, 60, 70, 80, 90,100, 125, 150, 175, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000,1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, ormore nucleotides, up to the entire length of the nucleic acid molecule.It is understood that for the purposes of determining sequence identitywhen comparing a DNA sequence to an RNA sequence, a thymine nucleotideis equivalent to an uracil nucleotide.

By “substantial identity” or “substantially identical” is meant apolypeptide or nucleic acid sequence that has the same polypeptide ornucleic acid sequence, respectively, as a reference sequence, or has aspecified percentage of amino acid residues or nucleotides,respectively, that are the same at the corresponding location within areference sequence when the two sequences are optimally aligned. Forexample, an amino acid sequence that is “substantially identical” to areference sequence has at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%,99%, or 100% sequence identity to the reference amino acid sequence. Forpolypeptides, the length of comparison sequences will generally be atleast 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 50,75, 90, 100, 150, 200, 250, 300, or 350 contiguous amino acids (e.g., afull-length sequence). For nucleic acids, the length of comparisonsequences will generally be at least 5, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, or 25 contiguous nucleotides (e.g., thefull-length nucleotide sequence). Sequence identity may be measuredusing sequence analysis software on the default setting (e.g., SequenceAnalysis Software Package of the Genetics Computer Group, University ofWisconsin Biotechnology Center, 1710 University Avenue, Madison, WI,53705). Such software may match similar sequences by assigning degreesof homology to various substitutions, deletions, and othermodifications.

The term “chimeric” as used herein as applied to a nucleic acid orpolypeptide refers to two components that are defined by structuresderived from different sources. For example, where “chimeric” is used inthe context of a chimeric polypeptide (e.g., a chimeric Cas9/Csn1protein), the chimeric polypeptide includes amino acid sequences thatare derived from different polypeptides. A chimeric polypeptide maycomprise either modified or naturally-occurring polypeptide sequences(e.g., a first amino acid sequence from a modified or unmodifiedCas9/Csn1 protein; and a second amino acid sequence other than theCas9/Csn1 protein). Similarly, “chimeric” in the context of apolynucleotide encoding a chimeric polypeptide includes nucleotidesequences derived from different coding regions (e.g., a firstnucleotide sequence encoding a modified or unmodified Cas9/Csn1 protein;and a second nucleotide sequence encoding a polypeptide other than aCas9/Csn1 protein).

The term “chimeric polypeptide” refers to a polypeptide that is made bythe combination (i.e., “fusion”) of two otherwise separated segments ofamino sequence, usually through human intervention. A polypeptide thatcomprises a chimeric amino acid sequence is a chimeric polypeptide.

Some chimeric polypeptides can be referred to as “fusion variants.”“Heterologous,” as used herein, means a nucleotide or polypeptidesequence that is not found in the native nucleic acid or protein,respectively. For example, in a chimeric Cas9/Csn1 protein, theRNA-binding domain of a naturally-occurring bacterial Cas9/Csn1polypeptide (or a variant thereof) may be fused to a heterologouspolypeptide sequence (i.e., a polypeptide sequence from a protein otherthan Cas9/Csn1 or a polypeptide sequence from another organism). Theheterologous polypeptide sequence may exhibit an activity (e.g.,enzymatic activity) that will also be exhibited by the chimericCas9/Csn1 protein (e.g., methyltransferase activity, acetyltransferaseactivity, kinase activity, ubiquitinating activity, etc.). Aheterologous nucleic acid sequence may be linked to anaturally-occurring nucleic acid sequence (or a variant thereof) (e.g.,by genetic engineering) to generate a chimeric nucleotide sequenceencoding a chimeric polypeptide. As another example, in a fusion variantCas9 site-directed polypeptide, a variant Cas9 site-directed polypeptidemay be fused to a heterologous polypeptide (i.e., a polypeptide otherthan Cas9), which exhibits an activity that will also be exhibited bythe fusion variant Cas9 site-directed polypeptide. A heterologousnucleic acid sequence may be linked to a variant Cas9 site-directedpolypeptide (e.g., by genetic engineering) to generate a nucleotidesequence encoding a fusion variant Cas9 site-directed polypeptide.

“Recombinant,” as used herein, means that a particular nucleic acid, asdefined herein, is the product of various combinations of cloning,restriction, polymerase chain reaction (PCR) and/or ligation stepsresulting in a construct having a structural coding or non-codingsequence distinguishable from endogenous nucleic acids found in naturalsystems. DNA sequences encoding polypeptides can be assembled from cDNAfragments or from a series of synthetic oligonucleotides, to provide asynthetic nucleic acid which is capable of being expressed from arecombinant transcriptional unit contained in a cell or in a cell-freetranscription and translation system. Genomic DNA comprising therelevant sequences can also be used in the formation of a recombinantgene or transcriptional unit. Sequences of non-translated DNA may bepresent 5′ or 3′ from the open reading frame, where such sequences donot interfere with manipulation or expression of the coding regions, andmay indeed act to modulate production of a desired product by variousmechanisms. Alternatively, DNA sequences encoding RNA (e.g.,DNA-targeting RNA) that is not translated may also be consideredrecombinant. Thus, e.g., the term “recombinant” nucleic acid refers toone which is not naturally occurring, e.g., is made by the artificialcombination of two otherwise separated segments of sequence throughhuman intervention. This artificial combination is often accomplished byeither chemical synthesis means, or by the artificial manipulation ofisolated segments of nucleic acids, e.g., by genetic engineeringtechniques. Such is usually done to replace a codon with a codonencoding the same amino acid, a conservative amino acid, or anon-conservative amino acid. Alternatively, it is performed to jointogether nucleic acid segments of desired functions to generate adesired combination of functions. This artificial combination is oftenaccomplished by either chemical synthesis means, or by the artificialmanipulation of isolated segments of nucleic acids, e.g., by geneticengineering techniques. When a recombinant polynucleotide encodes apolypeptide, the sequence of the encoded polypeptide can be naturallyoccurring (“wild type”) or can be a variant (e.g., a mutant) of thenaturally occurring sequence. Thus, the term “recombinant” polypeptidedoes not necessarily refer to a polypeptide whose sequence does notnaturally occur. Instead, a “recombinant” polypeptide is encoded by arecombinant DNA sequence, but the sequence of the polypeptide can benaturally occurring (“wild type”) or non-naturally occurring (e.g., avariant, a mutant, etc.). Thus, a “recombinant” polypeptide is theresult of human intervention, but may be a naturally occurring aminoacid sequence.

A “target sequence” as used herein is a polynucleotide (e.g., as definedherein, including a DNA, RNA, or DNA/RNA hybrid, as well as modifiedforms thereof) that includes a “target site.” The terms “target site” or“target protospacer DNA” are used interchangeably herein to refer to anucleic acid sequence present in a target genomic sequence (e.g., DNA orRNA in a host cell) to which a targeting portion of the guidingcomponent will bind provided sufficient conditions (e.g., sufficientcomplementarity) for binding exist. Suitable DNA/RNA binding conditionsinclude physiological conditions normally present in a cell. Othersuitable DNA/RNA binding conditions (e.g., conditions in a cell-freesystem) are known in the art; see, e.g., Sambrook, supra.

By “cleavage” it is meant the breakage of the covalent backbone of atarget sequence (e.g., a nucleic acid molecule). Cleavage can beinitiated by a variety of methods including, but not limited to,enzymatic or chemical hydrolysis of a phosphodiester bond. Bothsingle-stranded cleavage and double-stranded cleavage are possible, anddouble-stranded cleavage can occur as a result of two distinctsingle-stranded cleavage events. DNA cleavage can result in theproduction of either blunt ends or staggered ends. In certainembodiments, a complex comprising a guiding component and a nuclease isused for targeted double-stranded DNA cleavage. In other embodiments, acomplex comprising a guiding component and a nuclease is used fortargeted single-stranded RNA cleavage.

“Nuclease” and “endonuclease” are used interchangeably herein to mean anenzyme which possesses catalytic activity for DNA cleavage and/or RNAcleavage.

By “cleavage domain” or “active domain” or “nuclease domain” of anuclease it is meant the polypeptide sequence or domain within thenuclease which possesses the catalytic activity for nucleic acidcleavage. A cleavage domain can be contained in a single polypeptidechain or cleavage activity can result from the association of two (ormore) polypeptides. A single nuclease domain may consist of more thanone isolated stretch of amino acids within a given polypeptide.

A “host cell,” as used herein, denotes an in vivo or in vitro eukaryoticcell, a prokaryotic cell (e.g., bacterial or archaeal cell), or a cellfrom a multicellular organism (e.g., a cell line) cultured as aunicellular entity, which eukaryotic or prokaryotic cells can be, orhave been, used as recipients for a nucleic acid, and include theprogeny of the original cell which has been transformed by the nucleicacid. It is understood that the progeny of a single cell may notnecessarily be completely identical in morphology or in genomic or totalDNA complement as the original parent, due to natural, accidental, ordeliberate mutation. A “recombinant host cell” (also referred to as a“genetically modified host cell”) is a host cell into which has beenintroduced a heterologous nucleic acid, e.g., an expression vector. Forexample, a subject bacterial host cell is a genetically modifiedbacterial host cell by virtue of introduction into a suitable bacterialhost cell of an exogenous nucleic acid (e.g., a plasmid or recombinantexpression vector) and a subject eukaryotic host cell is a geneticallymodified eukaryotic host cell (e.g., a mammalian germ cell), by virtueof introduction into a suitable eukaryotic host cell of an exogenousnucleic acid.

By “linker” or “spacer”, unless otherwise indicated, is meant any usefulmultivalent (e.g., bivalent) component useful for joining to differentportions or segments. Exemplary linkers and spacers include a nucleicacid sequence, a chemical linker, etc. In one instance, the linker ofthe guiding component (e.g., linker L in the interacting portion of theguiding component) can have a length of from about 3 nucleotides toabout 100 nucleotides. For example, the linker can have a length of fromabout 3 nucleotides (nt) to about 90 nt, from about 3 nucleotides (nt)to about 80 nt, from about 3 nucleotides (nt) to about 70 nt, from about3 nucleotides (nt) to about 60 nt, from about 3 nucleotides (nt) toabout 50 nt, from about 3 nucleotides (nt) to about 40 nt, from about 3nucleotides (nt) to about 30 nt, from about 3 nucleotides (nt) to about20 nt or from about 3 nucleotides (nt) to about 10 nt. For example, thelinker can have a length of from about 3 nt to about 5 nt, from about 5nt to about 10 nt, from about 10 nt to about 15 nt, from about 15 nt toabout 20 nt, from about 20 nt to about 25 nt, from about 25 nt to about30 nt, from about 30 nt to about 35 nt, from about 35 nt to about 40 nt,from about 40 nt to about 50 nt, from about 50 nt to about 60 nt, fromabout 60 nt to about 70 nt, from about 70 nt to about 80 nt, from about80 nt to about 90 nt, or from about 90 nt to about 100 nt. In someembodiments, the linker of a single-molecule guiding component is 4 nt.

The term “histone-packaged supercoiled plasmid DNA” is used to describea component of particles according to the present disclosure that employa plasmid DNA that has been “supercoiled” (i.e., folded in on itselfusing a supersaturated salt solution or other ionic solution whichcauses the plasmid to fold in on itself and “supercoil” in order tobecome more dense for efficient packaging into the particles). Theplasmid may be virtually any plasmid that expresses any number ofpolypeptides or encode RNA, including small hairpin RNA/shRNA or smallinterfering RNA/siRNA, as otherwise described herein. Once supercoiled(using the concentrated salt or other anionic solution), the supercoiledplasmid DNA is then complexed with histone proteins to produce ahistone-packaged “complexed” supercoiled plasmid DNA.

“Packaged” DNA herein refers to DNA that is loaded into particles (e.g.,adsorbed into the pores, confined directly within the core itself, orconfined partially within a pore). To minimize the DNA spatially, it isoften packaged, which can be accomplished in several different ways,from adjusting the charge of the surrounding medium to creation of smallcomplexes of the DNA with, for example, lipids, proteins, or othernanoparticles (usually, although not exclusively cationic). Packaged DNAis often achieved via lipoplexes (i.e., complexing DNA with cationiclipid mixtures). In addition, DNA has also been packaged with cationicproteins (including proteins other than histones), as well as goldnanoparticles (e.g., NanoFlares—an engineered DNA and metal complex inwhich the core of the nanoparticle is gold).

One ore more histone proteins, as well as other means to package the DNAinto a smaller volume such as normally cationic nanoparticles, lipids,or proteins, may be used to package the supercoiled plasmid DNA“histone-packaged supercoiled plasmid DNA.” In certain aspects of thedisclosed technology, a combination of histone proteins H1, H2A, H2B,H3, and H4 in a preferred ratio of 1:2:2:2:2, although other histoneproteins may be used in other, similar ratios, as is known in the art ormay be readily practiced pursuant to the teachings of the presentdisclosure. The DNA may also be double stranded linear DNA, instead ofplasmid DNA, which also may be optionally supercoiled and/or packagedwith histones or other packaging components.

Other histone proteins which may be used in this aspect of the disclosedtechnology include, for example, H1F, H1A, H1B, H2A, H2B, H1F0, H1FNT,H1FOO, H1FX, H1H1, HIST1H1A, HIST1H1B, HIST1H1C, HIST1H1D, HIST1H1E,HIST1H1T, H2AF, H2AFB1, H2AFB2, H2AFB3, H2AFJ, H2AFV, H2AFX, H2AFY,H2AFY2, H2AFZ, H2A1, HIST1H2AA, HIST1H2AB, HIST1H2AC, HIST1H2AD,HIST1H2AE, HIST1H2AG, HIST1H2AI, HIST1H2AJ, HIST1H2AK, HIST1H2AL,HIST1H2AM, H2A2, HIST2H2AA3, HIST2H2AC, H2BF, H2BFM, HSBFS, HSBFWT,H2B1, HIST1H2BA, HIST1HSBB, HIST1HSBC, HIST1HSBD, HIST1H2BE, HIST1H2BF,HIST1H2BG, HIST1H2BH, HIST1H2B1, HIST1H2BJ, HIST1H2BK, HIST1H2BL,HIST1H2BM, HIST1H2BN, HIST1H2BO, H2B2, HIST2H2BE, H3A1, HIST1H3A,HIST1H3B, HIST1H3C, HIST1H3D, HIST1H3E, HIST1H3F, HIST1H3G, HIST1H3H,HIST1H3I, HIST1H3J, H3A2, HIST2H3C, H3A3, HIST3H3, H41, HIST1H4A,HIST1H4B, HIST1H4C, HIST1H4D, HIST1H4E, HIST1H4F, HIST1H4G, HIST1H4H,HIST1H4I, HIST1H4J, HIST1H4K, HIST1H4L, H44, and HIST4H4.

The term “nuclear localization sequence” refers to a peptide sequenceincorporated or otherwise crosslinked into histone proteins, whichcomprise the histone-packaged supercoiled plasmid DNA. In certainembodiments, particles according to the present disclosure may furthercomprise a plasmid (often a histone-packaged supercoiled plasmid DNA)which is modified (crosslinked) with a nuclear localization sequence(note that the histone proteins may be crosslinked with the nuclearlocalization sequence or the plasmid itself can be modified to express anuclear localization sequence), which enhances the ability of thehistone-packaged plasmid to penetrate the nucleus of a cell and depositits contents there (to facilitate expression and ultimately cell death.These peptide sequences assist in carrying the histone-packaged plasmidDNA and the associated histones into the nucleus of a targeted cell,whereupon the plasmid will express peptides and/or nucleotides asdesired to deliver therapeutic and/or diagnostic molecules (polypeptideand/or nucleotide) into the nucleus of the targeted cell. One or morecrosslinking agents, known in the art, may be used to covalently link anuclear localization sequence to a histone protein (often at a lysinegroup or other group which has a nucleophilic or electrophilic group inthe side chain of the amino acid exposed pendant to the polypeptide),which can be used to introduce the histone packaged plasmid into thenucleus of a cell. Alternatively, a nucleotide sequence that expressesthe nuclear localization sequence can be positioned in a plasmid inproximity to that which expresses histone protein, such that theexpression of the histone protein conjugated to the nuclear localizationsequence will occur thus facilitating transfer of a plasmid into thenucleus of a targeted cell.

The terms “nucleic acid regulatory sequences,” “control elements,” and“regulatory elements,” used interchangeably herein, refer totranscriptional and translational control sequences, such as promoters,enhancers, polyadenylation signals, internal ribosomal entry sites(IRES), terminators, and protein degradation signals, that provide forand/or regulate transcription of a non-coding sequence (e.g.,DNA-targeting RNA) or a coding sequence (e.g., site-directed modifyingpolypeptide, or Cas9/Csn1 polypeptide) and/or regulate translation of anencoded polypeptide.

A “promoter sequence” is a DNA regulatory region capable of binding RNApolymerase in a cell and initiating transcription of a downstream (3′direction) coding sequence. For purposes of defining the presentdisclosed technology, the promoter sequence is bounded at its 3′terminus by the transcription initiation site and extends upstream (5′direction) to include the minimum number of bases or elements necessaryto initiate transcription at levels detectable above background. Withinthe promoter sequence will be found a transcription initiation, as wellas protein binding domains (consensus sequences) responsible for thebinding of RNA polymerase. Eukaryotic promoters will often, but notalways, contain “TATA” boxes and “CAT” boxes. Prokaryotic promoterscontain Shine-Dalgarno sequences in addition to the −10 and −35consensus sequences.

An “expression control sequence” is a DNA sequence that controls andregulates the transcription and translation of another DNA sequence. Acoding sequence is “under the control” of transcriptional andtranslational control sequences in a cell when RNA polymerasetranscribes the coding sequence into mRNA, which is then translated intothe protein encoded by the coding sequence. Transcriptional andtranslational control sequences are DNA regulatory sequences, such aspromoters, enhancers, polyadenylation signals, and terminators, thatprovide for the expression of a coding sequence in a host cell.

A “vector” or “expression vector” is a replicon, such as a plasmid,phage, virus, or cosmid, to which another nucleic acid segment, i.e., an“insert”, may be attached so as to bring about the replication of theattached segment in a cell.

An “expression cassette” comprises a nucleic acid coding sequenceoperably linked, as defined herein, to a promoter sequence, as definedherein.

A “signal sequence” can be included before the coding sequence. Thissequence encodes a signal peptide, N-terminal to the polypeptide, thatcommunicates to the host cell to direct the polypeptide to the cellsurface or secrete the polypeptide into the media, and this signalpeptide is clipped off by the host cell before the protein leaves thecell. Signal sequences can be found associated with a variety ofproteins native to prokaryotes and eukaryotes.

“Operably linked” or “operatively linked” or “operatively associatedwith,” as used interchangeably, refers to a juxtaposition wherein thecomponents so described are in a relationship permitting them tofunction in their intended manner. For instance, a promoter is operablylinked to a coding sequence if the promoter affects its transcription orexpression. A nucleic acid molecule is operatively linked or operablylinked to, or operably associated with, an expression control sequencewhen the expression control sequence controls and regulates thetranscription and translation of nucleic acid sequence. The term“operatively linked” includes having an appropriate start signal (e.g.,ATG) in front of the nucleic acid sequence to be expressed andmaintaining the correct reading frame to permit expression of thenucleic acid sequence under the control of the expression controlsequence and production of the desired product encoded by the nucleicacid sequence. If a gene that one desires to insert into a recombinantDNA molecule does not contain an appropriate start signal, such a startsignal can be inserted in front of the gene.

In accordance with the present disclosure there may be employedconventional molecular biology, microbiology, and recombinant DNAtechniques within the skill of the art. Such techniques are explainedfully in the literature. See, e.g., Sambrook et al, 2001, “MolecularCloning: A Laboratory Manual”; Ausubel, ed., 1994, “Current Protocols inMolecular Biology” Volumes I-III; Celis, ed., 1994, “Cell Biology: ALaboratory Handbook” Volumes I-III; Coligan, ed., 1994, “CurrentProtocols in Immunology” Volumes I-III; Gait ed., 1984, “OligonucleotideSynthesis”; Hames & Higgins eds., 1985, “Nucleic Acid Hybridization”;Hames & Higgins, eds., 1984, “Transcription And Translation”; Freshney,ed., 1986, “Animal Cell Culture”; IRL.

By an “effective amount” or a “sufficient amount” of an agent (e.g., acargo), as used herein, is that amount sufficient to effect beneficialor desired results, such as clinical results, and, as such, an“effective amount” depends upon the context in which it is beingapplied. For example, in the context of administering an agent thatemploys a CRISPR component to genetically modify a gene, an effectiveamount of an agent is, for example, an amount sufficient to achieveincreased or decreased expression of that gene, as compared to theresponse obtained without administration of the agent.

By “subject” is meant a human or non-human animal (e.g., a mammal).

By “treating” a disease, disorder, or condition in a subject is meantreducing at least one symptom of the disease, disorder, or condition byadministrating a therapeutic agent to the subject. By “treatingprophylactically” a disease, disorder, or condition in a subject ismeant reducing the frequency of occurrence of or reducing the severityof a disease, disorder or condition by administering a therapeutic agentto the subject prior to the onset of disease symptoms. Beneficial ordesired results can include, but are not limited to, alleviation oramelioration of one or more symptoms or conditions; diminishment ofextent of disease, disorder, or condition; stabilized (i.e., notworsening) state of disease, disorder, or condition; preventing spreadof disease, disorder, or condition; delay or slowing the progress of thedisease, disorder, or condition; amelioration or palliation of thedisease, disorder, or condition; and remission (whether partial ortotal), whether detectable or undetectable.

By “salt” is meant an ionic form of a compound or structure (e.g., anyformulas, compounds, or compositions described herein), which includes acation or anion compound to form an electrically neutral compound orstructure. For example, non-toxic salts are described in Berge S M etal., “Pharmaceutical salts,” J. Pharm. Sci. 1977 January; 66(1):1-19;and in “Handbook of Pharmaceutical Salts: Properties, Selection, andUse,” Wiley-VCH, April 2011 (2nd rev. ed., eds. P. H. Stahl and C. G.Wermuth). The salts can be prepared in situ during the final isolationand purification of the compounds of the disclosed technology orseparately by reacting the free base group with a suitable organic acid(thereby producing an anionic salt) or by reacting the acid group with asuitable metal or organic salt (thereby producing a cationic salt).Representative anionic salts include acetate, adipate, alginate,ascorbate, aspartate, benzenesulfonate, benzoate, bicarbonate,bisulfate, bitartrate, borate, bromide, butyrate, camphorate,camphorsulfonate, chloride, citrate, cyclopentanepropionate,digluconate, dihydrochloride, diphosphate, dodecylsulfate, edetate,ethanesulfonate, fumarate, glucoheptonate, glucomate, glutamate,glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide,hydrochloride, hydroiodide, hydroxyethanesulfonate, hydroxynaphthoate,iodide, lactate, lactobionate, laurate, lauryl sulfate, malate, maleate,malonate, mandelate, mesylate, methanesulfonate, methylbromide,methylnitrate, methylsulfate, mucate, 2-naphthalenesulfonate,nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate,persulfate, 3-phenylpropionate, phosphate, picrate, pivalate,polygalacturonate, propionate, salicylate, stearate, subacetate,succinate, sulfate, tannate, tartrate, theophyllinate, thiocyanate,triethiodide, toluenesulfonate, undecanoate, and valerate salts.Representative cationic salts include metal salts, such as alkali oralkaline earth salts, e.g., barium, calcium (e.g., calcium edetate),lithium, magnesium, potassium, and sodium; other metal salts, such asaluminum, bismuth, iron, and zinc; as well as nontoxic ammonium,quaternary ammonium, and amine cations, including, but not limited toammonium, tetramethylammonium, tetraethylammonium, methylamine,dimethylamine, trimethylamine, triethylamine, ethylamine, andpyridinium. Other cationic salts include organic salts, such aschloroprocaine, choline, dibenzylethylenediamine, diethanolamine,ethylenediamine, methylglucamine, and procaine. Exemplary salts includepharmaceutically acceptable salts.

By “pharmaceutically acceptable salt” is meant a salt that is, withinthe scope of sound medical judgment, suitable for use in contact withthe tissues of humans and animals without for example, undue toxicity,irritation, or allergic response, and are commensurate with a reasonablebenefit/risk ratio.

By “pharmaceutically acceptable excipient” is meant any ingredient otherthan a compound or structure (e.g., any formulas, compounds, orcompositions described herein) and having the properties of beingnontoxic and non-inflammatory in a subject. Exemplary, non-limitingexcipients include adjuvants, antiadherents, antioxidants, binders,carriers, coatings, compression aids, diluents, disintegrants,dispersing agents, dyes (colors), emollients, emulsifiers, fillers(diluents), film formers or coatings, flavors, fragrances, glidants(flow enhancers), isotonic carriers, lubricants, preservatives, printinginks, solvents, sorbents, stabilizers, suspensing or dispersing agents,surfactants, sweeteners, waters of hydration, or wetting agents. Any ofthe excipients can be selected from those approved, for example, by theUnited States Food and Drug Administration or other governmental agencyas being acceptable for use in humans or domestic animals. Exemplaryexcipients include, but are not limited to alcohol, butylatedhydroxytoluene (BHT), calcium carbonate, calcium phosphate (dibasic),calcium stearate, croscarmellose, cross-linked polyvinyl pyrrolidone,citric acid, crospovidone, cysteine, ethylcellulose, gelatin, glycerol,hydroxypropyl cellulose, hydroxypropyl methylcellulose, lactatedRinger's solution, lactose, magnesium stearate, maltitol, maltose,mannitol, methionine, methylcellulose, methyl paraben, microcrystallinecellulose, polyethylene glycol, polyol, polyvinyl pyrrolidone, povidone,pregelatinized starch, propyl paraben, retinyl palmitate, Ringer'ssolution, shellac, silicon dioxide, sodium carboxymethyl cellulose,sodium chloride injection, sodium citrate, sodium starch glycolate,sorbitol, starch (corn), stearic acid, stearic acid, sucrose, talc,titanium dioxide, vegetable oil, vitamin A, vitamin E, vitamin C, water,and xylitol.

As used herein, the terms “top,” “bottom,” “upper,” “lower,” “above,”and “below” are used to provide a relative relationship betweenstructures. The use of these terms does not indicate or require that aparticular structure must be located at a particular location in theapparatus.

By “alkenyl” is meant an optionally substituted C₂₋₂₄ alkyl group havingone or more double bonds. The alkenyl group can be cyclic (e.g., C₃₋₂₄cycloalkenyl) or acyclic. The alkenyl group can also be substituted orunsubstituted. For example, the alkenyl group can be substituted withone or more substitution groups, as described herein for alkyl.

By “alkyl” and the prefix “alk” is meant a branched or unbranchedsaturated hydrocarbon group of 1 to 24 carbon atoms, such as methyl,ethyl, n-propyl, isopropyl, n-butyl, isobutyl, s-butyl, t-butyl,n-pentyl, isopentyl, s-pentyl, neopentyl, hexyl, heptyl, octyl, nonyl,decyl, dodecyl, tetradecyl, hexadecyl, eicosyl, and tetracosyl. Thealkyl group can be cyclic (e.g., C₃₋₂₄ cycloalkyl) or acyclic.

The alkyl group can be branched or unbranched. The alkyl group can alsobe substituted or unsubstituted. For example, the alkyl group can besubstituted with one, two, three or, in the case of alkyl groups of twocarbons or more, four substituents independently selected from the groupconsisting of: (1) C₁₋₆ alkoxy (e.g., —OAk, in which Ak is an alkylgroup, as defined herein); (2) C₁₋₆ alkylsulfinyl (e.g., —S(O)Ak, inwhich Ak is an alkyl group, as defined herein); (3) C₁₋₆ alkylsulfonyl(e.g., —SO₂Ak, in which Ak is an alkyl group, as defined herein); (4)amino (e.g., —NR^(N1)R^(N2), where each of R^(N1) and R^(N2) is,independently, H or optionally substituted alkyl, or R^(N1) and R^(N2),taken together with the nitrogen atom to which each are attached, form aheterocyclyl group); (5) aryl; (6) arylalkoxy (e.g., —OA^(L)Ar, in whichA^(L) is an alkylene group and Ar is an aryl group, as defined herein);(7) aryloyl (e.g., —C(O)Ar, in which Ar is an aryl group, as definedherein); (8) azido (e.g., an —N₃ group); (9) cyano (e.g., a —CN group);(10) carboxyaldehyde (e.g., a —C(O)H group); (11) C₃₋₈ cycloalkyl; (12)halo; (13) heterocyclyl (e.g., a 5-, 6- or 7-membered ring, unlessotherwise specified, containing one, two, three, or four non-carbonheteroatoms (e.g., independently selected from the group consisting ofnitrogen, oxygen, phosphorous, sulfur, or halo)); (14) heterocyclyloxy(e.g., —OHet, in which Het is a heterocyclyl group); (15)heterocyclyloyl (e.g., —C(O)Het, in which Het is a heterocyclyl group);(16) hydroxyl (e.g., a —OH group); (17) N-protected amino; (18) nitro(e.g., an —NO₂ group); (19) oxo (e.g., an ═O group); (20) C₃₋₈spirocyclyl (e.g., an alkylene diradical, both ends of which are bondedto the same carbon atom of the parent group to form a spirocyclylgroup); (21) C₁₋₆ thioalkoxy (e.g., —SAk, in which Ak is an alkyl group,as defined herein); (22) thiol (e.g., an —SH group); (23) —CO₂R^(A),where R^(A) is selected from the group consisting of (a) hydrogen, (b)C₁₋₆ alkyl, (c) C₄₋₁₈ aryl, and (d) C₁₋₆ alk-C₄₋₁₈ aryl; (24)—C(O)NR^(B)R^(C), where each of R^(B) and R^(C) is, independently,selected from the group consisting of (a) hydrogen, (b) C₁₋₆ alkyl, (c)C₄₋₁₈ aryl, and (d) C₁₋₆ alk-C₄₋₁₈ aryl; (25) —SO₂R^(D), where R^(D) isselected from the group consisting of (a) C₁₋₆ alkyl, (b) C₄₋₁₈ aryl,and (c) C₁₋₆ alk-C₄₋₁₈ aryl; (26) —SO₂NR^(E)R^(F), where each of R^(E)and R^(F) is, independently, selected from the group consisting of (a)hydrogen, (b) C₁₋₆ alkyl, (c) C₄₋₁₈ aryl, and (d) C₁₋₆ alk-C₄₋₁₈ aryl;and (27) —NR^(G)R^(H), where each of R^(G) and R^(H) is, independently,selected from the group consisting of (a) hydrogen, (b) an N-protectinggroup, (c) C₁₋₆ alkyl, (d) C₂₋₆ alkenyl, (e) C₂₋₆ alkynyl, (f) C₄₋₁₈aryl, (g) C₁₋₆ alk-C₄₋₁₈ aryl, (h) C₃₋₈ cycloalkyl, and (i) C₁₋₆alk-C₃₋₈ cycloalkyl, wherein in one embodiment no two groups are boundto the nitrogen atom through a carbonyl group or a sulfonyl group. Thealkyl group can be a primary, secondary, or tertiary alkyl groupsubstituted with one or more substituents (e.g., one or more halo oralkoxy). In some embodiments, the unsubstituted alkyl group is a C₁₋₃,C₁₋₆, C₁₋₁₂, C₁₋₁₆, C₁₋₁₈, C₁₋₂₀, or C₁₋₂₄ alkyl group.

By “alkylene” is meant a multivalent (e.g., bivalent, trivalent,tetravalent, etc.) form of an alkyl group, as described herein.Exemplary alkylene groups include methylene, ethylene, propylene,butylene, etc. In some embodiments, the alkylene group is a C₁₋₃, C₁₋₆,C₁₋₁₂, C₁₋₁₆, C₁₋₁₈, C₁₋₂₀, C₁₋₂₄, C₂₋₃, C₂₋₆, C₂₋₁₂, C₂₋₁₆, C₂₋₁₈,C₂₋₂₀, or C₂₋₂₄ alkylene group. The alkylene group can be branched orunbranched. The alkylene group can also be substituted or unsubstituted.For example, the alkylene group can be substituted with one or moresubstitution groups, as described herein for alkyl.

By “alkynyl” is meant an optionally substituted C₂₋₂₄ alkyl group havingone or more triple bonds. The alkynyl group can be cyclic or acyclicsuch as ethynyl or 1-propynyl. The alkynyl group can also be substitutedor unsubstituted. For example, the alkynyl group can be substituted withone or more substitution groups, as described herein for alkyl.

By “amido” is meant —C(O)NR^(N1)R^(N2), where each of R^(N1) and R^(N2)is, independently, H, optionally substituted alkyl, or optionallysubstituted aryl; or where a combination of R^(N1) and R^(N2), takentogether with the nitrogen atom to which each are attached, form aheterocyclyl group, as defined herein.

By “amino” is meant —NR^(N1)R^(N2), where each of R^(N1) and R^(N2) is,independently, H or optionally substituted alkyl, or R^(N1) and R^(N2),taken together with the nitrogen atom to which each are attached, form aheterocyclyl group, as defined herein.

By “aryl” is meant a group that contains any carbon-based aromatic groupincluding, but not limited to, benzyl, naphthalene, phenyl, biphenyl,and phenoxybenzene. The term “aryl” also includes “heteroaryl,” which isdefined as a group that contains an aromatic group that has at least oneheteroatom incorporated within the ring of the aromatic group. Examplesof heteroatoms include, but are not limited to, nitrogen, oxygen,sulfur, and phosphorus. Likewise, the term “non-heteroaryl,” which isalso included in the term “aryl,” defines a group that contains anaromatic group that does not contain a heteroatom. The aryl group can besubstituted or unsubstituted. The aryl group can be substituted withone, two, three, four, or five substituents independently selected fromthe group consisting of: (1) C₁₋₆ alkanoyl (e.g., —C(O)Ak, in which Akis an alkyl group, as defined herein); (2) C₁₋₆ alkyl; (3) C₁₋₆ alkoxy(e.g., —OAk, in which Ak is an alkyl group, as defined herein); (4) C₁₋₆alkoxy-C₁₋₆ alkyl (e.g., an alkyl group, which is substituted with analkoxy group —OAk, in which Ak is an alkyl group, as defined herein);(5) C₁₋₆ alkylsulfinyl (e.g., —S(O)Ak, in which Ak is an alkyl group, asdefined herein); (6) C₁₋₆ alkylsulfinyl-C₁₋₆ alkyl (e.g., an alkylgroup, which is substituted by an alkylsulfinyl group —S(O)Ak, in whichAk is an alkyl group, as defined herein); (7) C₁₋₆ alkylsulfonyl (e.g.,—SO₂Ak, in which Ak is an alkyl group, as defined herein); (8) C₁₋₆alkylsulfonyl-C₁₋₆ alkyl (e.g., an alkyl group, which is substituted byan alkylsulfonyl group —SO₂Ak, in which Ak is an alkyl group, as definedherein); (9) aryl; (10) amino (e.g., —NR^(N1)R^(N2), where each ofR^(N1) and R^(N2) is, independently, H or optionally substituted alkyl,or R^(N1) and R^(N2), taken together with the nitrogen atom to whicheach are attached, form a heterocyclyl group); (11) C₁₋₆ aminoalkyl(e.g., meant an alkyl group, as defined herein, substituted by an aminogroup); (12) heteroaryl; (13) C₁₋₆ alk-C₄₋₁₈ aryl (e.g., -A^(L)Ar, inwhich A^(L) is an alkylene group and Ar is an aryl group, as definedherein); (14) aryloyl (e.g., —C(O)Ar, in which Ar is an aryl group, asdefined herein); (15) azido (e.g., an —N3 group); (16) cyano (e.g., a—CN group); (17) C₁₋₆ azidoalkyl (e.g., a —N3 azido group attached tothe parent molecular group through an alkyl group, as defined herein);(18) carboxyaldehyde (e.g., a —C(O)H group); (19) carboxyaldehyde-C₁₋₆alkyl (e.g., -A^(L)C(O)H, in which A^(L) is an alkylene group, asdefined herein); (20) C₃₋₈ cycloalkyl; (21) C₁₋₆ alk-C₃₋₈ cycloalkyl(e.g., -A^(L)Cy, in which A^(L) is an alkylene group and Cy is acycloalkyl group, as defined herein); (22) halo (e.g., F, Cl, Br, or I);(23) C₁₋₆ haloalkyl (e.g., an alkyl group, as defined herein,substituted with one or more halo); (24) heterocyclyl; (25)heterocyclyloxy (e.g., —OHet, in which Het is a heterocyclyl group);(26) heterocyclyloyl (e.g., —C(O)Het, in which Het is a heterocyclylgroup); (16) hydroxyl (e.g., a —OH group); (27) hydroxyl (e.g., a —OHgroup); (28) C₁₋₆ hydroxyalkyl (e.g., an alkyl group, as defined herein,substituted by one to three hydroxyl groups, with the proviso that nomore than one hydroxyl group may be attached to a single carbon atom ofthe alkyl group); (29) nitro (e.g., an —NO₂ group); (30) C₁₋₆ nitroalkyl(e.g., an alkyl group, as defined herein, substituted by one to threenitro groups); (31)N-protected amino; (32) N-protected amino-C₁₋₆ alkyl;(33) oxo (e.g., an ═O group); (34) C₁₋₆ thioalkoxy (e.g., —SAk, in whichAk is an alkyl group, as defined herein); (35) thio-C₁₋₆ alkoxy-C₁₋₆alkyl (e.g., an alkyl group, which is substituted by an thioalkoxy group—SAk, in which Ak is an alkyl group, as defined herein); (36)—(CH₂)_(r)CO₂R^(A), where r is an integer of from zero to four, andR^(A) is selected from the group consisting of (a) hydrogen, (b) C₁₋₆alkyl, (c) C₄₋₁₈ aryl, and (d) C₁₋₆ alk-C₄₋₁₈ aryl; (37)—(CH₂)_(r)CONR^(B)R^(C), where r is an integer of from zero to four andwhere each R^(B) and R^(C) is independently selected from the groupconsisting of (a) hydrogen, (b) C₁₋₆ alkyl, (c) C₄₋₁₈ aryl, and (d) C₁₋₆alk-C₄₋₁₈ aryl; (38) —(CH₂)_(r)SO₂R^(D), where r is an integer of fromzero to four and where R^(D) is selected from the group consisting of(a) C₁₋₆ alkyl, (b) C₄₋₁₈ aryl, and (c) C₁₋₆ alk-C₄₋₁₈ aryl; (39)—(CH₂)_(r)SO₂NR^(E)R^(F), where r is an integer of from zero to four andwhere each of R^(E) and R^(F) is, independently, selected from the groupconsisting of (a) hydrogen, (b) C₁₋₆ alkyl, (c) C₄₋₁₈ aryl, and (d) C₁₋₆alk-C₄₋₁₈ aryl; (40) —(CH₂)_(r)NR^(G)R^(H), where r is an integer offrom zero to four and where each of R^(G) and R^(H) is, independently,selected from the group consisting of (a) hydrogen, (b) an N-protectinggroup, (c) C₁₋₆ alkyl, (d) C₂₋₆ alkenyl, (e) C₂₋₆ alkynyl, (f) C₄₋₁₈aryl, (g) C₁₋₆ alk-C₄₋₁₈ aryl, (h) C₃₋₈ cycloalkyl, and (i) C₁₋₆alk-C₃₋₈ cycloalkyl, wherein in one embodiment no two groups are boundto the nitrogen atom through a carbonyl group or a sulfonyl group; (41)thiol; (42) perfluoroalkyl (e.g., an alkyl group, as defined herein,having each hydrogen atom substituted with a fluorine atom); (43)perfluoroalkoxy (e.g., —ORf, in which Rf is an alkyl group, as definedherein, having each hydrogen atom substituted with a fluorine atom);(44) aryloxy (e.g., —OAr, where Ar is an optionally substituted arylgroup, as described herein); (45) cycloalkoxy (e.g., —OCy, in which Cyis a cycloalkyl group, as defined herein); (46) cycloalkylalkoxy (e.g.,—OA^(L)Cy, in which A^(L) is an alkylene group and Cy is a cycloalkylgroup, as defined herein); and (47) arylalkoxy (e.g., —OA^(L)Ar, inwhich A^(L) is an alkylene group and Ar is an aryl group, as definedherein). In particular embodiments, an unsubstituted aryl group is aC₄₋₁₈, C₄₋₁₄, C₄₋₁₂, C₄₋₁₀, C₆₋₁₈, C₆₋₁₄, C₆₋₁₂, or C₆₋₁₀ aryl group.

By “arylene” is meant a multivalent (e.g., bivalent, trivalent,tetravalent, etc.) form of an aryl group, as described herein. Exemplaryarylene groups include phenylene, naphthylene, biphenylene,triphenylene, diphenyl ether, acenaphthenylene, anthrylene, orphenanthrylene. In some embodiments, the arylene group is a C₄₋₁₈,C₄₋₁₄, C₄₋₁₂, C₄₋₁₀, C₆₋₁₈, C₆₋₁₄, C₆₋₁₂, or C₆₋₁₀ arylene group. Thearylene group can be branched or unbranched. The arylene group can alsobe substituted or unsubstituted. For example, the arylene group can besubstituted with one or more substitution groups, as described hereinfor aryl.

By “azido” is meant an —N₃ group.

By “carbamido” is meant —NR^(N1)C(O)NR^(N2)R^(N3), where each of R^(N1)and R^(N2) and R^(N3) is, independently, H, optionally substitutedalkyl, or optionally substituted aryl; or where a combination of R^(N2)and R^(N3), taken together with the nitrogen atom to which each areattached, form a heterocyclyl group, as defined herein.

By “carbonyl” is meant a —C(O)— group, which can also be represented as>C═O.

By “carboxyl” is meant a —CO₂H group.

By “halo” is meant F, Cl, Br, or I.

By “heteroalkyl” is meant an alkyl group, as defined herein, containingone, two, three, or four non-carbon heteroatoms (e.g., independentlyselected from the group consisting of nitrogen, oxygen, phosphorous,sulfur, or halo).

By “heteroalkylene” is meant a divalent form of an alkylene group, asdefined herein, containing one, two, three, or four non-carbonheteroatoms (e.g., independently selected from the group consisting ofnitrogen, oxygen, phosphorous, sulfur, or halo).

By “heteroaryl” is meant a subset of heterocyclyl groups, as definedherein, which are aromatic, i.e., they contain 4n+2 pi electrons withinthe mono- or multicyclic ring system.

By “heterocyclyl” is meant a 5-, 6- or 7-membered ring, unless otherwisespecified, containing one, two, three, or four non-carbon heteroatoms(e.g., independently selected from the group consisting of nitrogen,oxygen, phosphorous, sulfur, or halo). The 5-membered ring has zero totwo double bonds and the 6- and 7-membered rings have zero to threedouble bonds. The term “heterocyclyl” also includes bicyclic, tricyclicand tetracyclic groups in which any of the above heterocyclic rings isfused to one, two, or three rings independently selected from the groupconsisting of an aryl ring, a cyclohexane ring, a cyclohexene ring, acyclopentane ring, a cyclopentene ring, and another monocyclicheterocyclic ring, such as indolyl, quinolyl, isoquinolyl,tetrahydroquinolyl, benzofuryl, and benzothieny. Heterocyclics include,for example, thiiranyl, thietanyl, tetrahydrothienyl, thianyl,thiepanyl, aziridinyl, azetidinyl, pyrrolidinyl, piperidinyl, azepanyl,pyrrolyl, pyrrolinyl, pyrazolyl, pyrazolinyl, pyrazolidinyl, imidazolyl,imidazolinyl, imidazolidinyl, pyridyl, homopiperidinyl, pyrazinyl,piperazinyl, pyrimidinyl, pyridazinyl, oxazolyl, oxazolidinyl,isoxazolyl, isoxazolidiniyl, morpholinyl, thiomorpholinyl, thiazolyl,thiazolidinyl, isothiazolyl, isothiazolidinyl, indolyl, quinolinyl,isoquinolinyl, benzimidazolyl, benzothiazolyl, benzoxazolyl, furyl,thienyl, thiazolidinyl, isothiazolyl, isoindazoyl, triazolyl,tetrazolyl, oxadiazolyl, uricyl, thiadiazolyl, pyrimidyl,tetrahydrofuranyl, dihydrofuranyl, tetrahydrothienyl, dihydrothienyl,dihydroindolyl, tetrahydroquinolyl, tetrahydroisoquinolyl, pyranyl,dihydropyranyl, dithiazolyl, benzofuranyl, and benzothienyl.

By “hydroxyl” is meant —OH.

By “imido” is meant —C(O)N^(N1)C(O)—, where R^(N1) is, independently, H,optionally substituted alkyl, or optionally substituted aryl.

By “protecting group” is meant any group intended to protect a reactivegroup against undesirable synthetic reactions. Commonly used protectinggroups are disclosed in “Greene's Protective Groups in OrganicSynthesis,” John Wiley & Sons, New York, 2007 (4th ed., eds. P. G. M.Wuts and T. W. Greene), which is incorporated herein by reference.O-protecting groups include an optionally substituted alkyl group (e.g.,forming an ether with reactive group O), such as methyl, methoxymethyl,methylthiomethyl, benzoyloxymethyl, t-butoxymethyl, etc.; an optionallysubstituted alkanoyl group (e.g., forming an ester with the reactivegroup O), such as formyl, acetyl, chloroacetyl, fluoroacetyl (e.g.,perfluoroacetyl), methoxyacetyl, pivaloyl, t-butylacetyl, phenoxyacetyl,etc.; an optionally substituted aryloyl group (e.g., forming an esterwith the reactive group O), such as —C(O)—Ar, including benzoyl; anoptionally substituted alkylsulfonyl group (e.g., forming analkylsulfonate with reactive group O), such as —SO₂—R^(S1), where R^(S1)is optionally substituted C₁₋₁₂ alkyl, such as mesyl or benzylsulfonyl;an optionally substituted arylsulfonyl group (e.g., forming anarylsulfonate with reactive group O), such as —SO₂—R^(S4), where R^(S4)is optionally substituted C₄₋₁₈ aryl, such as tosyl or phenylsulfonyl;an optionally substituted alkoxycarbonyl or aryloxycarbonyl group (e.g.,forming a carbonate with reactive group O), such as —C(O)—OR^(T1), whereR^(T1) is optionally substituted C₁₋₁₂ alkyl or optionally substitutedC₄₋₁₈ aryl, such as methoxycarbonyl, methoxymethylcarbonyl,t-butyloxycarbonyl (Boc), or benzyloxycarbonyl (Cbz); or an optionallysubstituted silyl group (e.g., forming a silyl ether with reactive groupO), such as —Si—(R^(T2))₃, where each R^(T2) is, independently,optionally substituted C₁₋₁₂ alkyl or optionally substituted C₄₋₁₈ aryl,such as trimethylsilyl, t-butyldimethylsilyl, or t-butyldiphenylsilyl.N-protecting groups include, e.g., formyl, acetyl, benzoyl, pivaloyl,t-butylacetyl, alanyl, phenylsulfonyl, benzyl, Boc, and Cbz. Suchprotecting groups can employ any useful agent to cleave the protectinggroup, thereby restoring the reactivity of the unprotected reactivegroup.

By “thio” is meant an —S— group.

By “thiol” is meant an —SH group.

By “attaching,” “attachment,” or related word forms is meant anycovalent or non-covalent bonding interaction between two components.Non-covalent bonding interactions include, without limitation, hydrogenbonding, ionic interactions, halogen bonding, electrostaticinteractions, π bond interactions, hydrophobic interactions, inclusioncomplexes, clathration, van der Waals interactions, and combinationsthereof.

Other features and advantages of the disclosed technology will beapparent from the following description and the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-1C is a schematic showing exemplary constructs. Provided are (A)an exemplary method 100 for providing a non-limiting construct having acore 101, a spacer 103, a cargo 104, and an outer layer 105; (B) anotherexemplary construct 1000; and (C) yet another exemplary construct 1500.

FIG. 2 is a schematic showing an exemplary lipid coated mesoporoussilica nanoparticles (LC-MSN) in accordance with present disclosure.

FIG. 3A-3C is a schematic showing exemplary methods for providing aconstruct and its use for in vitro gene editing.

FIG. 4A-4E is a schematic view showing (A) exemplary linking agents andmethods employing linking agent L1 to provide a construct having ananoparticle (NP) core, a spacer, and an attached cargo, (B) anotherexemplary spacer having a reactive group L” that interacts with areactive group R¹ present on a cargo, and yet other exemplary spacerspresent between the NP and the cargo. Also provided are (D) anotherexemplary spacer and (B) another exemplary method employing linkingagent L2 to provide a construct having a NP core, a spacer, and anattached cargo.

FIG. 5A-5C is a schematic view showing further exemplary linking agentsand spacers. Provided are schematics of (A) exemplary spacers (i)-(iii)present between a core and a cargo, (B) an exemplary reaction schemebetween a linking agent and a reactive group present on a cargo, therebyforming a spacer between the core and the cargo, and (C) yet anotherreaction scheme between another linking agent and a reactive grouppresent on a cargo.

FIG. 6 is a schematic view of an exemplary CRISPR component thatincludes a guiding component 90 to bind to the target sequence 97, aswell as a nuclease 98.

FIG. 7A-7C are schematics showing exemplary CRISPR components.

FIG. 8A-8H shows non-limiting amino acid sequences for variousnucleases. Provided are sequences for (A) a Cas9 endonuclease for S.pyogenes serotype M1 (SEQ ID NO:110), (B) a deactivated Cas9 having D10Aand H840A mutations (SEQ ID NO:111), (C) a Cas protein Csn1 for S.pyogenes (SEQ ID NO:112), (D) a Cas9 endonuclease for F. novicida U112(SEQ ID NO:113), (E) a Cas9 endonuclease for S. thermophilus 1 (SEQ IDNO:114), (F) a Cas9 endonuclease for S. thermophilus 2 (SEQ ID NO:115),(G) a Cas9 endonuclease for L. innocua (SEQ ID NO:116), and (H) a Cas9endonuclease for W succinogenes (SEQ ID NO:117).

FIG. 9 shows non-limiting nucleic acid sequences of crRNA that can beemployed as a first portion in any guiding component described herein.Provided are sequences for S. pyogenes (SEQ ID NO:20), L. innocua (SEQID NO:21), S. thermophilus 1 (SEQ ID NO:22), S. thermophilus 2 (SEQ IDNO:23), F. novicida (SEQ ID NO:24), and W. succinogenes (SEQ ID NO:25).Also provided are various consensus sequences (SEQ ID NOs:26-32), inwhich each X, independently, can be absent, A, C, T, G, or U, as well asmodified forms thereof (e.g., as described herein). In anotherembodiment, for each consensus sequence (SEQ ID NOs:26-32), each X ateach position is a nucleic acid (or a modified form thereof) that isprovided in an aligned reference sequence. For instance, for consensusSEQ ID NO:26, the first position includes an X, and this X can be absentor any nucleic acid (e.g., A, C, T, G, or U, as well as modified formsthereof). Alternatively, this X can be any nucleic acid provided in analigned reference sequence (e.g., aligned reference sequences SEQ IDNO:20-25 for the consensus sequence in SEQ ID NO:26). Thus, X atposition 1 in SEQ ID NO:26 can also be G (as in SEQ ID NOs:20-23 and 25)or C (as in SEQ ID NO:24), in which this subset of substitutions isdefined as a conservative subset. Similarly, for each X at each positionfor the consensus sequences (SEQ ID NOs:26-32), conservative subsets canbe determined based on FIG. 9 , and these consensus sequences includenucleic acid sequences encompassed by such conservative subsets. Grayhighlight indicates a conserved nucleic acid, and the dash indicates anabsent nucleic acid.

FIG. 10A-10C shows non-limiting nucleic acid sequences of tracrRNA thatcan be employed as a second portion and/or linker in any guidingcomponent described herein. Provided are sequences for S. pyogenes (SEQID NO:40), L. innocua (SEQ ID NO:41), S. thermophilus 1 (SEQ ID NO:42),S. thermophilus 2 (SEQ ID NO:43), F. novicida 1 (SEQ ID NO:44), F.novicida 2 (SEQ ID NO:45), W. succinogenes 1 (SEQ ID NO:46), and Wsuccinogenes 2 (SEQ ID NO:47). Also provided are various consensussequences (SEQ ID NOs:48-54), in which each Z, independently, can beabsent, A, C, T, G, or U, as well as modified forms thereof (e.g., asdescribed herein). Consensus sequences are shown for (A) an alignment ofall SEQ ID NOs:40-47, providing consensus sequences SEQ ID NOs:48-50;(B) an alignment of SEQ ID NOs:40-43, providing consensus sequences SEQID NOs:51-52; and (C) an alignment of SEQ ID NOs:44-47, providingconsensus sequences SEQ ID NOs:53-54. In another embodiment, for eachconsensus sequence (SEQ ID NOs:48-54), each Z at each position is anucleic acid (or a modified form thereof) that is provided in an alignedreference sequence. For instance, for consensus SEQ ID NO:48, the firstposition includes a Z, and this Z can be absent or any nucleic acid(e.g., A, C, T, G, or U, as well as modified forms thereof).Alternatively, this Z can be any nucleic acid provided in an alignedreference sequence (e.g., aligned reference sequences SEQ ID NO:40-47for the consensus sequence in SEQ ID NO:48). Thus, Z at position 2 inSEQ ID NO:48 can also be U (as in SEQ ID NOs:40, 41, and 43-47) or G (asin SEQ ID NO:42), in which this subset of substitutions is defined as aconservative subset. Similarly, for each Z at each position for theconsensus sequences (SEQ ID NOs:48-54), conservative subsets can bedetermined based on FIG. 10A-10C, and these consensus sequences includenucleic acid sequences encompassed by such conservative subsets. Grayhighlight indicates a conserved nucleic acid, and the dash indicates anabsent nucleic acid.

FIG. 17 shows non-limiting nucleic acid sequences of extended tracrRNAthat can be employed as a second portion and/or linker in any guidingcomponent described herein. Provided are sequences for S. pyogenes (SEQID NO:60), L. innocua (SEQ ID NO:61), S. thermophilus 1 (SEQ ID NO:62),and S. thermophilus 2 (SEQ ID NO:63). Also provided are variousconsensus sequences (SEQ ID NOs:64-65), in which each Z, independently,can be absent, A, C, T, G, or U, as well as modified forms thereof(e.g., as described herein). In another embodiment, for each consensussequence (SEQ ID NOs:64-65), each Z at each position is a nucleic acid(or a modified form thereof) that is provided in an aligned referencesequence. For instance, for consensus SEQ ID NO:64, the first positionincludes a Z, and this Z can be absent or any nucleic acid (e.g., A, C,T, G, or U, as well as modified forms thereof). Alternatively, this Zcan be any nucleic acid provided in an aligned reference sequence (e.g.,aligned reference sequences SEQ ID NO:60-63 for the consensus sequencein SEQ ID NO:64). Thus, Z at position 1 in SEQ ID NO:64 can also beabsent (as in SEQ ID NO:60), A (as in SEQ ID NO:61), or U (as in SEQ IDNOs:63-64), in which this subset of substitutions is defined as aconservative subset. Similarly, for each Z at each position for theconsensus sequences (SEQ ID NOs:64-65), conservative subsets can bedetermined based on FIG. 17 , and these consensus sequences includenucleic acid sequences encompassed by such conservative subsets. Grayhighlight indicates a conserved nucleic acid, and the dash indicates anabsent nucleic acid.

FIG. 12 shows non-limiting nucleic acid sequences of a guiding component(e.g., a synthetic, non-naturally occurring guiding component) having ageneric structure of A-L-B, in which A includes a first portion (e.g.,any one of SEQ ID NOs:20-32, or a fragment thereof), L is a linker(e.g., a covalent bond, a nucleic acid sequence, a fragment of any oneof SEQ ID NOs:40-54 and 60-65, or any other useful linker), and B is asecond portion (e.g., any one of SEQ ID NOs:40-54 and 60-65, or afragment thereof). Also provided are various embodiments ofsingle-stranded guiding components (SEQ ID NOs:80-93). Exemplarynon-limiting guiding components include SEQ ID NO:81, or a fragmentthereof, where X at each position is defined as in SEQ ID NO:26 and Z ateach position is as defined in SEQ ID NO:48; SEQ ID NO:82, or a fragmentthereof, where X at each position is defined as in SEQ ID NO:27 and Z ateach position is as defined in SEQ ID NO:49; SEQ ID NO:83, where X ateach position is defined as in SEQ ID NO:28 and Z at each position is asdefined in SEQ ID NO:49; SEQ ID NO:84, or a fragment thereof, where X ateach position is defined as in SEQ ID NO:27 and Z at each position is asdefined in SEQ ID NO:65; SEQ ID NO:85, or a fragment thereof, where X ateach position is defined as in SEQ ID NO:28 and Z at each position is asdefined in SEQ ID NO:65; SEQ ID NO:86, or a fragment thereof, where X ateach position is defined as in SEQ ID NO:29 and Z at each position isdefined as in SEQ ID NO:51; SEQ ID NO:87, or a fragment thereof, where Xat each position is defined as in SEQ ID NO:30 and Z at each position isdefined as in SEQ ID NO:51; SEQ ID NO:88, or a fragment thereof, where Xat each position is defined as in SEQ ID NO:30 and Z at each position isdefined as in SEQ ID NO:52; SEQ ID NO:89, or a fragment thereof, where Xat each position is defined as in SEQ ID NO:30 and Z at each position isdefined as in SEQ ID NO:65; SEQ ID NO:90, or a fragment thereof, where Xat each position is defined as in SEQ ID NO:31 and Z at each position isdefined as in SEQ ID NO:51; SEQ ID NO:91, or a fragment thereof, where Xat each position is defined as in SEQ ID NO:32 and Z at each position isas defined in SEQ ID NO:53; SEQ ID NO:92, or a fragment thereof, where Xat each position is defined as in SEQ ID NO:32 and Z at each position isas defined in SEQ ID NO:54; and SEQ ID NO:93, or a fragment thereof,where X at each position is defined as in SEQ ID NO:32 and Z at eachposition is defined as in SEQ ID NO:65. The fragment can include anyuseful number of nucleotides (e.g., one or more contiguous nucleotides,such as a fragment including about 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 18,20, or more contiguous nucleotides of any sequences described herein,such as a sequence for the first portion, e.g., any one of SEQ IDNOs:20-32; and also such as a fragment including about 4, 5, 6, 7, 8, 9,10, 11, 12, 15, 18, 20, 24, 26, 28, 30, 32, 34, 38, 36, 40, or morecontiguous nucleotides of any sequences described herein, such as asequence for the first portion, e.g., any one of SEQ ID NOs:40-54 and60-65).

FIG. 13 shows additional non-limiting nucleic acid sequences of aguiding component (e.g., a synthetic, non-naturally occurring guidingcomponent). Provided are various embodiments of single-stranded guidingcomponents (SEQ ID NOs:100-103). Exemplary non-limiting guidingcomponents include SEQ ID NO:100, or a fragment thereof, where n at eachof positions 1-80 can be present or absent such that this region cancontain anywhere from 12 to 80 nucleotides and n is A, C, T, G, U, ormodified forms thereof; and where n at each of positions 93-192 can bepresent or absent such that this region can contain anywhere from 3 to100 nucleotides and n is A, C, T, G, U, or modified forms thereof; SEQID NO:101, or a fragment thereof, where n at each of positions 1-80 canbe present or absent such that this region can contain anywhere from 12to 80 nucleotides and n is A, C, T, G, U, or modified forms thereof; andwhere n at each of positions 93-192 can be present or absent such thatthis region can contain anywhere from 3 to 100 nucleotides and n is A,C, T, G, U, or modified forms thereof; SEQ ID NO:102, or a fragmentthereof, where n at each of positions 1-80 can be present or absent suchthat this region can contain anywhere from 12 to 80 nucleotides and n isA, C, T, G, U, or modified forms thereof, and SEQ ID NO:103, or afragment thereof, where n at each of positions 1-80 can be present orabsent such that this region can contain anywhere from 12 to 80nucleotides and n is A, C, T, G, U, or modified forms thereof.

FIG. 14A-C are TEM micrographs showing exemplary stellate nanoparticlesin various levels of magnification.

FIG. 15 is a graph showing porosimetry data.

FIG. 16 is a graph showing Cas9 loading and release in exemplary LC-MSNsby densitometry.

FIG. 17A-C are graphs showing editing efficiency of exemplary loadedLC-MSNs and comparative examples.

FIG. 18A-D are graphs showing size, charge, polydispersity, andcolloidal stability information of exemplary particles and comparativeexamples.

FIG. 19 is a schematic showing the mechanism of Example 5.

DETAILED DESCRIPTION

The present disclosure relates to particle-based constructs configuredto transport a CRISPR-based cargo in vivo and in vitro as an attempt toovercome a major hurdle in clinical translation of CRISPR-basedcountermeasures. The construct is directed to attacking viral infectionsby targeting critical host factor or viral genomes directly. Inparticular embodiments, the construct includes a porous nanoparticlecore, in which the pores are employed to completely or partially confinethe cargo. Nanoparticles of various pore sizes were screened todetermine those most effective in vitro editing efficiency, stability,and monodispersity. The construct and cargo are encapsulated partiallyor completely by a coating comprising a cationic lipid, a pegylatedlipid, a zwitterionic lipid, and a sterol.

The construct can include any useful component and can be assembled inany useful process. FIG. 1A provides an exemplary method 100 forassembling a construct. As can be seen, the method can include providinga core 101 including a plurality of pores 102. The pores are inside orpart of the external surface. Furthermore, a first pore can optionallybe connected to or coupled to a second pore. The pores can becharacterized in any useful manner, such as, e.g., by an averagedimension of the plurality of pores.

Optionally, the method can include expanding the pores present in thecore. In this instance, the pores of the initial core can becharacterized by a first dimension. After expansion, the initial corecan include a plurality of expanded pores, where an average dimension ofthe plurality of expanded pores is characterized by a second dimensionof the same type (e.g. length, width, or height) that is greater thanthe first dimension. Pore expansion can be accomplished in any usefulmanner, e.g., by use of a swelling agent to expand an initial pore sizeto a larger pore size.

Spacers can optionally be used to attach a cargo to the core. As seen inFIG. 1A, the method can optionally include installing 110 a spacer 103to be disposed within at least one pore and/or upon the external surfaceof the core. The spacer can be installed by use of a linking agent(e.g., L¹-R^(L)-L², in which R^(L) is a linking group such as anydescribed herein; each of L¹ and L² is, independently, a reactive groupsuch as any functional group described herein; and each of L¹ and L² canbe the same or different). The linking agent can include a firstreactive group to form a bond with the core, as well as a secondreactive group to form a bond with the cargo. The linking agent may bedivalent (having two reactive groups) or multivalent (having more thantwo reactive groups). If a linker is not used, certain cargo can beloaded through electrostatic interacations.

The cargo can be introduced to the core to provide a loaded core. Asseen in FIG. 1A, the method can include binding 120 a cargo 104 to aspacer. In one non-limiting instance, the installed spacer 103 caninclude a reactive group that interacts with a reactive group present onthe cargo, thereby forming a bond (e.g., a covalent or non-covalentbond). In other embodiments, the cargo itself has a reactive group thatinteracts directly with the core surface (particle).

Then, an outer layer 105 is provided on the external surface of the core130. The outer layer includes a blend of lipids and a sterol. In oneinstance, the method includes providing an outer layer 130, therebyforming an exemplary construct. The outer layer 105 can be formed, e.g.,by exposing the loaded core to a lipid and sterol formulation to formthe outer layer 105.

The outer layer can include one or more moieties (e.g., targetingligands). These moieties can be introduced before or after providing theouter layer. The cargo should be loaded before the outer layer. In yetother embodiments, the moieties can be introduced simultaneously withproviding the outer layer. In forming the outer layer 105, a lipidformulation including cationic and anionic lipids, the sterol components(e.g., cholesterol), and the targeting ligand moieties can be prepared;and the resulting formulation can be used to form the outer layer. Asseen in FIG. 1A, the method can includes providing 140 one or moremoieties 106, thereby forming an exemplary construct.

As seen in FIG. 1A, one exemplary construct includes a core 101 having aplurality of pores 102, a spacer 103 disposed within a pore, a cargo 104attached to the spacer, and an outer layer 105 optionally including amoiety 106. FIG. 1B provides another exemplary construct 1000 havinginterconnected pores 1002 within the core 1001, spacers 1003 disposed onan external surface of the core, a cargo 1004 attached to the spacer,and an outer layer 1005. In another instance, FIG. 1C provides yetanother exemplary construct 1500 having interconnected pores 1502 withinthe core 1501, spacers 1503 disposed on an external surface of the coreor within a pore, a cargo 1504 attached to the spacer, and an outerlayer 1505.

A particular embodiment with a highly effective outer layer is shown inFIG. 2 . The mesoporous nanoparticle 202 has a CRISPR-Cas9 cargo 204disposed in its pores, and the core is at least partially encapsulatedby an outer coating 206 comprising four components, a cationic lipid,DOTAP 208, a sterol, cholesterol 210, a zwitterionic lipid DOPE 212, anda Pegylated lipid, DSPE-PEG2000 214. A PEG portion 216 of theDSPE-PEG2000 component 214 extends outward from the encapsulating outercoating 206. The Examples below shows the efficient delivery ofCRISPR-56 Cas9 RNP components using lipid coated mesoporous silicananoparticles (LC-MSN) modified from previous studies.

Core

In an embodiment, the core is a particle, providing a surface upon whichan outer layer and a cargo can be supported. In other non-limitingembodiments, the core provides a charged surface that allows forelectrostatic interactions with the cargo and/or the outer layer, or aportion thereof.

In one instance, the core can be characterized by a first dimension(e.g., core circumference, pore size of the core, core diameter, corelength, or core width). Exemplary values for a core dimension (e.g.,core circumference, core diameter, core length, or core width, as wellas an average or mean value for any of these) include, withoutlimitation, greater than about 1 nm (e.g., greater than about 5 nm, 10nm, 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 125nm, 150 nm, 200 nm, 300 nm, 500 nm, 750 nm, 1 m, 2 m, 5 m, 10 m, or 20m), including from about 5 nm to about 300 nm (e.g., from 5 nm to 20 nm,5 nm to 30 nm, 5 nm to 40 nm, 5 nm to 50 nm, 5 nm to 75 nm, 5 nm to 100nm, 5 nm to 150 nm, 5 nm to 200 nm, 5 nm to 250 nm, 10 nm to 20 nm, 10nm to 30 nm, 10 nm to 40 nm, 10 nm to 50 nm, 10 nm to 75 nm, 10 nm to100 nm, 10 nm to 150 nm, 10 nm to 200 nm, 10 nm to 250 nm, 10 nm to 300nm, 25 nm to 30 nm, 25 nm to 40 nm, 25 nm to 50 nm, 25 nm to 75 nm, 25nm to 100 nm, 25 nm to 150 nm, 25 nm to 200 nm, 25 nm to 250 nm, 25 nmto 300 nm, 50 nm to 75 nm, 50 nm to 100 nm, 50 nm to 150 nm, 50 nm to200 nm, 50 nm to 250 nm, 50 nm to 300 nm, 75 nm to 100 nm, 75 nm to 150nm, 75 nm to 200 nm, 75 nm to 250 nm, 75 nm to 300 nm, 100 nm to 150 nm,100 nm to 200 nm, 100 nm to 250 nm, 100 nm to 300 nm, 150 nm to 200 nm,150 nm to 250 nm, 150 nm to 300 nm, 200 nm to 250 nm, 200 nm to 300 nm,250 nm to 300 nm, or 275 nm to 300 nm).

In one instance, the particle includes a porous core (e.g., a silicacore that is spherical and ranges in diameter from about 10 nm to about250 nm (e.g., having a mean diameter of about 150 nm)). In particularembodiments, the silica core is monodisperse in size distribution.

The core can be further characterized by an electrostatic property. Insome embodiments, the core has a negative charge (e.g., a net negativecharge), such as a zeta potential of from about −10 mV to about −200 mV(e.g., from −10 mV to −100 mV, −10 mV to −75 mV, −10 mV to −50 mV, −10mV to −30 mV, −15 mV to −100 mV, −15 mV to −75 mV, −15 mV to −50 mV, −15mV to −30 mV, −20 mV to −100 mV, −20 mV to −75 mV, −20 mV to −50 mV, −20mV to −30 mV, −30 mV to −100 mV, −30 mV to −75 mV, −30 mV to −50 mV, −40mV to −100 mV, −40 mV to −75 mV, −40 mV to −50 mV, −50 mV to −100 mV,−50 mV to −75 mV, −60 mV to −100 mV, or −60 mV to −75 mV). Zetapotential measurements are obtained using a Malvern Zetasizer.

The core can be porous. In particular embodiments, the pore has adimension (e.g., average pore size, pore diameter, pore radius, porecircumference, pore length, pore width, or pore depth) that is greaterthan about 0.5 nm (e.g., of from about 0.5 nm to about 30 nm, includingfrom 0.5 nm to 10 nm, 0.5 nm to 20 nm, 0.5 nm to 25 nm, 1 nm to 10 nm, 1nm to 15 nm, 1 nm to 20 nm, 1 nm to 25 nm, 1 nm to 30 nm, 2 nm to 5 nm,2 nm to 10 nm, 2 nm to 20 nm, 2 nm to 25 nm, or 2 nm to 30 nm).

A particle or a portion thereof (e.g., a core) may have a variety ofshapes and cross-sectional geometries that may depend, in part, upon theprocess used to produce the particles. The core or particle can be ananoparticle (e.g., having a diameter less than about 1 m) or amicroparticle (e.g., having a diameter greater than or equal to about 1m). In one embodiment, a core or particle may have a shape that is asphere, a donut (toroidal), a rod, a tube, a flake, a fiber, a plate, awire, a cube, or a whisker. A collection of cores may have two or moreof the aforementioned shapes. In one embodiment, a cross-sectionalgeometry of the core may be one or more of circular, ellipsoidal,triangular, rectangular, or polygonal. In one embodiment, a core mayconsist essentially of non-spherical cores. For example, such cores mayhave the form of ellipsoids, which may have all three principal axes ofdiffering lengths, or may be oblate or prelate ellipsoids of revolution.Non-spherical cores alternatively may be laminar in form, whereinlaminar refers to particles in which the maximum dimension along oneaxis is substantially less than the maximum dimension along each of theother two axes. Non-spherical cores may also have the shape of frusta ofpyramids or cones, or of elongated rods. In one embodiment, the coresmay be irregular in shape. In one embodiment, a plurality of cores mayconsist essentially of spherical cores. Particles and cores for use inthe present disclosure may be PEGylated and/or aminated as otherwisedescribed in Int. Pub. Nos. WO 2015/042268 and WO 2015/042279, which isincorporated herein by reference in their entirety.

The particle size distribution (e.g., size of the core for the protocellor a size of the silica carrier), according to the present disclosure,depends on the application, but is principally monodisperse (e.g., auniform sized population varying no more than about 5-20% in diameter,as otherwise described herein). In certain embodiments, particles orcores can range, e.g., from around 1 nm to around 500 nm in size,including all integers and ranges there between. The size is measured asthe longest axis of the core. In various embodiments, the cores are fromaround 5 nm to around 500 nm and from around 10 nm to around 100 nm insize. In certain alternative embodiments, the cores or particles aremonodisperse and range in size from about 25 nm to about 300 nm. Thesizes used include 50 nm (+/−10 nm) and 150 nm (+/−15 nm), within anarrow monodisperse range, but may be more narrow in range.

When the core is porous, the pores can be from around 0.5 nm to about 25nm in diameter, often about 1 to around 20 nm in diameter, including allintegers and ranges there between. In one embodiment, the pores are fromaround 1 to around 10 nm in diameter. In one embodiment, around 90% ofthe pores are from around 1 to around 20 nm in diameter. In anotherembodiment, around 95% of the pores are around 1 to around 20 nm indiameter.

In certain embodiments, cores or particles according to the disclosedtechnology: are monodisperse and range in size from about 25 nm to about300 nm; are anionic, neutral or cationic for specific targeting (e.g,cationic); are optionally modified with agents such as PEI (polyethyleneimine), NMe³⁺, dye, crosslinker, ligands (ligands provide neutralcharge); and optionally, are used in combination with a cargo to bedelivered to the target.

In some embodiments, these cores or particles are often monodisperse andprovide colloidally stable compositions. These compositions can be usedto target host cells because of enhanced biodistribution/bioavailabilityof these compositions, and optionally, specific cells, with a widevariety of therapeutic and/or diagnostic agents that exhibit varyingrelease rates at the site of activity.

The cores can be produced, for example, by templating with a surfactant,a cross-linked micelle, or a detergent (see, e.g., Gao F et al., J Phys.Chem. B. 2009; 113:1796-804; Lin Y S et al., Chem. Mater. 2009;21(17):3979-86; Carroll N J et al., Langmuir 2009; 25(23):13540-4; andZhang K et al., J. Am. Chem. Soc. 2013 Feb. 20; 135(7):2427-30). In yetanother instance, cores are formed by dendritic growth (see, e.g., ShenD et al., Nano Lett. 2014; 14(2):923-32). In some instances, the coresare formed by expanding a pore, e.g., by use of a swelling agent, suchas an alkylbenzene (e.g., 1,3,5-trimethylbenzene ortriisopropylbenzene), an alkane (e.g., heptane, decane, or dodecane), aglycol (e.g., poly(propylene glycol)), or a tertiary amine (see, e.g.,Kim M H et al., ACS Nano 2011; 5(5):3568-76; and Na H K et al., Small2012; 8(11):1752-61). In other instances, cores are formed by an aerosolprocess, such as EISA (see, e.g., Lu Y et al., Nature 1999; 398:223-6;and Durfee P N et al., ACS Nano 2016; 10:8325-45).

Each batch of cores or particles can be characterized by, for example,assessment of size and polydispersity using dynamic light scattering(DLS) (NIST-NCL PCC-1), and surface charge or zeta potentialmeasurements (e.g., with a Zetasizer instrument (Malvern Instruments,Ltd) (NIST-NCL PCC-2 (charge and zeta potential), and verification oflow endotoxin contamination per health industry product standards (NCLSTE-1.1). Resultant cores can be further processed, such as by modifyingcore condensation (e.g., by using acidified ethanol for silica) ormodifying core surface charge (e.g., by use of amine-containing silanes,such as APTES).

The core can be formed of, for example, a metal oxide, alum, or silica,including mesoporous forms thereof). In particular embodiments, the coreis composed of a mesoporous silica nanoparticle (MSN). Exemplary,non-limiting MSNs for use in the disclosed technology are described inInt. Pub. Nos. WO 2015/042268 and WO 2015/042279, each of which isincorporated herein in its entirety.

In an embodiment, a stellate particle is used as the core. A stellateparticle has a radial pore morphology with a small particle diameter. Italso has more uniform polydispersity (FIGS. 18A and 18C) and colloidalstability (FIG. 18D) compared to other particle types such as small-porehexagonal prism, dendritic MSN with 8 or 18 nm pores, and non-porousStober MSN. FIG. 18D shows comparison of the RNP-loaded MSN with andwithout lipids. FIG. 18C shows size measurements during LC-MSN assembly.The FIG. 18B illustrates the overall charge of the particle. Neutrallycharged particles are more stable in vivo than charged particles. Thecombination of the negatively charged nanoparticle cores (MSN) with thepositively charged lipid (cationic lipid), has a neutral charge ifparticles are unloaded (LC-MSN) or even when loaded with RNP (RNP-LC-MSNand 488-RNP-LC-MSN). The negatively charged core and positively charged(cationic) lipid promotes in vivo stability and delivery efficacy.

The synthesis of stellate MSN involves a base-catalyzed condensationreaction of tetraorthosilicate with a surfactant that acts as asubstrate. A single synthesis reaction can yield between 500-800milligrams of MSN that are stable when stored in ethanol for over ayear.

DLS and TEM were used to assess the size and morphology of stellate MSN(FIG. 14A-C). The unique radiating arm morphology of the stellateparticles can be seen in FIGS. 14A-C. The average size (diameter) ofstellate particles may be, for example, about 75 to 400 nm, such asabout 100 to 200 nm, or about 110 to 160 nm. Arm length for the stellateparticle may range from about 30 to 200 nm, such as about 50 to 100 nm,or about 55 to 80 nm. The average diameter may be assessed by aZetasizer instrument (Malvern Instruments, Ltd). Porosimetry wasperformed with nitrogen adsorption-desorption analysis to determine thepore size range, which may be, for example, 3 to 20 nm, such as 5 to 15nm or 6.5 to 10 nm (FIG. 15 ).

While MSNs are promising therapeutic carriers, MSNs can have lowcolloidal stability and are subject to aggregation in physiologicalsolutions, reducing circulation time and preventing desirable celluptake. Similarly, permanently charged cationic liposomes aresuccessfully used as nucleic acid transfection reagents in cell culture,however, they have limited in vivo stability. The net neutral charge(FIG. 18B) of the RNP loaded LC-MSNs overcomes challenges presented bythese individual components to enable improvements in colloidalstability and subsequent circulation time, with biocompatibility andlower cytoxicity.

In an embodiment, a spacer can be employed to attach a core (e.g., anexternal surface and/or a pore of the core) to one or more cargos. Aspacer can include, for example, a bond (e.g., a covalent bond or acoordination bond), an atom, a molecule, a nucleic acid, or a protein. Aspacer can be provided as a linking agent, which in turn reacts with areactive group (e.g., a functional group present on the core or thecargo) to form a bond. Thus, a reacted linking agent can result in aspacer present between the core and the cargo.

A spacer can include a coordination bond. In some instances, thecoordination bond includes one or more functional groups that form abond to a metal (e.g., a divalent metal). Exemplary functional groupsinclude an amino, an amido, a carboxyl, a thiol, a heterocyclyl (e.g., aheteroaryl, imidazolyl, etc.), or an amino acid (e.g., histidine,cysteine, lysine, etc.), as well as chelate forms thereof (e.g., as iniminodiacetic acid or nitriloacetic acid). Exemplary metals includenickel, cobalt, copper, iron, or zinc, as well as cationic formsthereof.

A non-zero length spacer can include a linking group. In some instances,a linking agent (e.g., to form the non-zero length spacer) includes atleast two reactive groups and a linking group disposed between thereactive groups. In some instances, a first reactive group forms a bondwith the core, and a second reactive group forms a bond with the cargo.The linking group can be, for example, an optionally substitutedalkylene, heteroalkylene, arylene, nucleic acid, or peptide, and canhave a functionality, such as, for example, a cleavable moiety, therebydetaching the cargo from the core.

The spacer can optionally include a cleavable moiety. Exemplarycleavable moieties include a labile group, or a scissile group,including but not limited to a disulfide bond.

The spacer can be provided as a linking agent, which in turn reacts witha reactive group (e.g., a functional group present on the core or thecargo) to form a bond. In some instances, the linking agent isL¹-R^(L)-L², in which R^(L) is a linking group (e.g., any usefulchemical group, such as a covalent bond, a nucleic acid sequence, amonomer, etc.) and each of L¹ and L² is, independently, a reactive group(e.g., a functional group that is one of a cross-linker group, a bindinggroup, or a click-chemistry group, such as any described herein), and inwhich each of L¹ and L² can be the same or different.

FIG. 4A provides an exemplary linking agent L^(1′)-Lk-L^(1″) (compoundL1), where Lk is a linking group and where each of L^(1′) and L^(1″) is,independently, a reactive group (e.g., a functional group that is one ofa cross-linker group, a binding group, or a click-chemistry group, suchas any described herein). In some instances, a reactive group caninclude a protecting group (e.g., any described herein), which providesa reactive group upon exposure to particular chemical or biologicalconditions (e.g., an acidic condition, a basic condition, the presenceof a protease, etc.).

As seen in FIG. 4A, a first group of the linking agent can be used toreact with a core (NP), thereby providing a NP-spacer. A second group ofthe linking agent can then react with a functional group R¹ of thecargo, thereby providing a NP-spacer-cargo construct. Any useful linkingagent and spacer can be employed.

FIG. 4B provided an exemplary spacer present between a core (NP) and thecargo. The spacer -Lk-L″*-R*, in which Lk is a linking group (e.g., anydescribed herein), L″* is a reactive group of the linking agent (thatunderwent a reaction), and R¹ is a second reactive group present on thecargo (that underwent a reaction). The dashed line indicates that thebond can be covalent or non-covalent.

Further spacers are provided in FIG. 4C. In some embodiments, the spacercan include a covalent bond between reacted L″*and reacted X. In oneinstance, L″*-X can be represented by a reacted sulfone group of thelinking agent, a reacted Lys of the cargo, and an alkylene group betweenthe sulfone and Lys. In another instance, two groups on the cargo reactwith the linking agent, thereby providing a multivalent spacerL″*<(His)₂ between the NP and the cargo. In other embodiments, thespacer can include a non-covalent bond between reacted L″* and reactedX. In one instance, L″*-X can be represented by a chelated Ni²⁺ of thelinking agent, a chelated His of the cargo, and a coordination bondbetween the nickel and His. In another instance, the linking agentprovides a reactive group L*, the cargo includes a reactive His, and anintermediate Ni²⁺ is provided to provide a chelating bridge between thelinking agent and the cargo.

FIG. 4D provided another exemplary spacer present between a core (NP)and the cargo. The spacer -Lk¹-L^(C)-Lk²-L″*-R¹*, in which Lk¹ is afirst linking group (e.g., any described herein), L^(C) is a cleavablemoiety (e.g., any described herein), Lk² is a second linking group(e.g., any described herein), L″* is a reactive group of the linkingagent (that underwent a reaction), and R¹* is a second reactive grouppresent on the cargo (that underwent a reaction). The dashed lineindicates that the bond can be covalent or non-covalent. Exemplarylinking groups (e.g., for Lk¹ and/or Lk²) includes an optionallysubstituted alkylene group, an optionally substituted heteroalkylenegroup, or a poly(ethylene glycol) group.

A cleavable moiety (L^(C)) can include any useful moiety capable ofreleasing a bound cargo upon exposure to a particular cleaving conditionor cleaving agent. In one non-limiting embodiment, the cleavable moietyincludes a disulfide group (e.g., —S—S—), in which the cleavingcondition includes a reducing condition and the cleaving agent is areducing agent (e.g., dithiothreitol (DTT),tris(2-carboxyethyl)phosphine (TCEP), or(2S)-2-amino-1,4-dimercaptobutane (DTBA)). In another non-limitingembodiment, the cleavable moiety includes a hydrazone group(e.g., >C═N—NH—), in which the cleaving condition includes an acidiccondition and the cleaving agent is an acidic agent (e.g., an acidhaving a pH less than about 4.5).

A reactive linking group of the linking agent (L″ or L″*) can includeany useful moiety, such as one or more anionic moieties (e.g., achelating anionic moiety, such as a polycarboxylic acid, a carboxylicacid, a carbonate, etc.) and one or more cationic moieties (e.g., achelated cationic metal, such as a cationic transition metal, includingCo²⁺, N^(i2+), Fe²⁺, Cu²⁺, or Zn²⁺). Further exemplary anionic moietiescan include those having one or more carboxylic or carbonate moieties,such as iminodiacetic acid (IDA), nitrilotriacetic acid (NTA),ethylenediamine tetraacetic acid (EDTA), diethylenetriaminepentaaceticacid (DTPA), (ethylene glycol-bis(β-aminoethylether)-N,N,N′,N′-tetraacetic acid) (EGTA),(1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid) (BAPTA),carboxylmethylaspartate (CMA), as well as acidic and basic formsthereof.

A second reactive group present on the cargo (R¹ or R¹*) can include anyuseful moiety capable of forming a bond with the reactive linking groupof the linking agent. In one non-limiting embodiment, the reactivelinking group includes a cationic moiety, and the second reactive grouppresent on the cargo is a moiety capable of forming a bond with thecationic moiety. Exemplary second reactive groups include one or morehistidine residues located at any useful position of the cargo (e.g., atthe N-terminus or the C-terminus for a protein cargo).

FIG. 4E provides an exemplary linking agent L^(1′)-Lk¹-L^(C)-Lk²-L^(1″)(compound L2), where Lk¹ and Lk² are linking groups, where L^(C) is acleavable moiety, and where each of L^(1′) and L^(1″) is, independently,a reactive group (e.g., a functional group that is one of a cross-linkergroup, a binding group, or a click-chemistry group, such as anydescribed herein). A first group of the linking agent L2 can be used toreact with a core (NP), thereby providing a NP-spacer. A second group ofthe linking agent can then react with a functional group R¹ of thecargo, thereby providing a NP-spacer-cargo construct. Any useful linkingagent and spacer can be employed. Next, as the linker as a cleavablemoiety L^(C), the construct can be exposed to a cleaving agent thatreacts with L^(C) to provide a released particle (released NP) and areleased cargo.

FIG. 5A-5B provides schematics of exemplary reaction schemes for linkingagents and spacers. As seen in FIG. 5A, the spacer can include multiplecoordination bonds (i), multiple covalent bonds (ii), or a singlecovalent bond (iii) between the core and the cargo. Such spacers canemploy any useful linking agent and reaction schemes. FIG. 5B providesan exemplary reaction scheme in which the linking agent includes areactive group L″ having an alkene and a sulfone leaving group. Thereactive group R¹ of the cargo participates in an addition reaction withL″, thereby providing a single covalent bond present in the spacer.Then, a second reactive group R² of the cargo participated in anotheraddition reaction with the linking agent, thereby providing a secondcovalent bond present in the spacer. FIG. 5C provides an exemplaryreaction scheme in which the linking agent includes a reactive group L″having an alkene and a sulfone leaving group, and the reactive group R¹of the cargo participates in an addition reaction to provide a singlecovalent bond. Other exemplary spacers and linking agents are describedin Cong Y et al., Bioconjug. Chem. 2012; 23(2):248-63; Liberatore F A etal., Bioconjug. Chem. 1990; 1(1):36-50; Han D H et al., Nature Commun.2014; 5(5):5633; and Shen D et al., Nano Lett. 2014; 14(2):923-32, eachof which is incorporated herein by reference in its entirety.

Reactive groups can be present on any useful bonding components, such asspacers, linking agents, a surface of the core, and/or a cargo. Pairs ofreactive groups can be chosen to facilitate any useful reaction betweenany bonding components. In one instance, the first bonding componentincludes a nucleophilic reactive group (e.g., an amino group, a thiogroup, a hydroxyl group, an anion, etc.), and the second bondingcomponent includes an electrophilic reactive group (e.g., an alkenylgroup, an alkynyl group, a carbonyl group, an ester group, an imidogroup, an epoxide group, an amido group, a carbamido group, a cation,etc.).

Exemplary reactive groups include any chemical group configured to forma bond. In general, a first chemical group reacts with a second chemicalgroup to form a bond (e.g., a covalent bond), in which the first andsecond chemical groups form a reactive pair.

In one instance, the reactive group is a cross-linker group. In anothernon-limiting instance, the reactive pair is a cross-linker reactionpair, which includes a first cross-linker group and a secondcross-linker group that reacts with that first cross-linker group.Exemplary cross-linker groups and cross-linker reaction pairs includethose for forming a covalent bond between a carboxyl group (e.g., —CO₂H)and an amino group (e.g., —NH₂); or between an imido group (e.g.,maleimido or succinimido) and a thiol group (e.g., —SH); or between anepoxide group and a thiol group (e.g., —SH); or between an epoxide groupand an amino group (e.g., —NH₂); or between an ester group (e.g., —CO₂R,in which R is an organic moiety, such as optionally substituted alkyl,aryl, etc.) and an amino group (e.g., —NH₂); or between an carbamidogroup (e.g., —NHC(O)Het, where Het is a N-containing heterocyclyl) andan amino group (e.g., —NH₂); or between a phospho group (e.g.,—P(O)(OH)₂) and an amino group (e.g., —NH₂), such as1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) anddicyclohexylcarbodiimide (DCC), optionally used withN-hydroxysuccinimide (NHS) and/or N-hydroxysulfosuccinimide (sulfo-NHS).Other cross-linkers include those for forming a covalent bond between anamino group (e.g., —NH₂) and a thymine moiety, such assuccinimidyl-[4-(psoralen-8-yloxy)]-butyrate (SPB); a hydroxyl group(e.g., —OH) and a sulfur-containing group (e.g., free thiol, —SH,sulfhydryl, cysteine moiety, or mercapto group), such asp-maleimidophenyl isocyanate (PMPI); between an amino group (e.g., —NH₂)and a sulfur-containing group (e.g., free thiol, —SH, sulfhydryl,cysteine moiety, or mercapto group), such as succinimidyl4-(p-maleimidophenyl)butyrate (SMPB) and/or succinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC); between asulfur-containing group (e.g., free thiol, —SH, sulfhydryl, cysteinemoiety, or mercapto group) and a carbonyl group (e.g., an aldehydegroup, such as for an oxidized glycoprotein carbohydrate), such asN-beta-maleimidopropionic acid hydrazide-trifluoroacetic acid salt(BMPH), 3-(2-pyridyldithio)propionyl hydrazide (PDPH), and/or a3-(2-pyridyldithio)propionyl group (PDP); and between amaleimide-containing group and a sulfur-containing group (e.g., freethiol, —SH, sulfhydryl, cysteine moiety, or mercapto group). Yet othercross-linkers include those for forming a covalent bond between two ormore unsaturated hydrocarbon bonds, e.g., such as a reaction of forminga covalent bond between a first alkene group and a second alkene group.

In another instance, the reactive group is a binding group. In anothernon-limiting instance, the reactive pair is a binding reaction pair,which includes a first binding group and a second binding group thatreacts with that first binding group. Exemplary binding groups andbinding reaction pairs include those for forming a bond between biotinand avidin, biotin and streptavidin, biotin and neutravidin,desthiobiotin and avidin (or a derivative thereof, such as streptavidinor neutravidin), hapten and an antibody, an antigen and an antibody, aprimary antibody and a secondary antibody, and lectin and aglycoprotein.

In yet another instance, the reactive group is a click-chemistry group.In another non-limiting instance, the reactive pair is a click-chemistryreaction pair, which includes a first click-chemistry group and a secondclick-chemistry group that reacts with that first click-chemistry group.Exemplary click-chemistry groups include, e.g., a click-chemistry group,e.g., one of a click-chemistry reaction pair selected from the groupconsisting of a Huisgen 1,3-dipolar cycloaddition reaction between analkynyl group and an azido group to form a triazole-containing spacer; aDiels-Alder reaction between a diene having a 4π electron system (e.g.,an optionally substituted 1,3-unsaturated compound, such as optionallysubstituted 1,3-butadiene, 1-methoxy-3-trimethylsilyloxy-1,3-butadiene,cyclopentadiene, cyclohexadiene, or furan) and a dienophile orheterodienophile having a 2π electron system (e.g., an optionallysubstituted alkenyl group or an optionally substituted alkynyl group); aring opening reaction with a nucleophile and a strained heterocyclylelectrophile; and a splint ligation reaction with a phosphorothioategroup and an iodo group; and a reductive amination reaction with analdehyde group and an amino group.

Exemplary reactive groups include an amino (e.g., —NH₂), a thio (e.g., athioalkoxy group or a thiol group), a hydroxyl, an ester (e.g., anacrylate), an imido (e.g., a maleimido or a succinimido), an epoxide, anisocyanate, an isothiocyanate, an anhydride, an amido, a carbamido(e.g., a urea derivative), an azide, an optionally substituted alkynyl,or an optionally substituted alkenyl.

Exemplary linking groups include any moiety, including any usefulsubunit, which can be optionally repeated, that provides a spacer havingany useful property. Exemplary linking groups include a bond (e.g., acovalent bond), optionally substituted alkylene, optionally substitutedheteroalkylene (e.g., poly(ethylene glycol)), optionally substitutedarylene, and optionally substituted heteroarylene. Yet other exemplarylinking groups are those including an ethylene glycol group, e.g.,—OCH₂CH₂—, including a poly(ethylene glycol) (PEG) group—(OCH₂CH₂)_(n)—, a four-arm PEG group (such as C(CH₂O(CH₂CH₂O)_(n)—)₄ orC(CH₂O(CH₂CH₂O)_(n)CH₂—)₄ or C(CH₂O(CH₂CH₂O)_(n)CH₂CH₂—)₄ orC(CH₂O(CH₂CH₂O)_(n)CH₂CH₂NHC(O)CH₂CH₂)₄ C(CH₂O(CH₂CH₂O)_(n)CH₂C(O)O—)₄),an eight-arm PEG group (such as —(OCH₂CH₂)_(n)O[CH₂CHO((CH₂CH₂O)_(n)—)CH₂O]₆(CH₂CH₂O)_(n) or—CH₂(OCH₂CH₂)_(n)O[CH₂CHO((CH₂CH₂O)_(n) CH₂)CH₂O]₆(CH₂CH₂O)_(n)CH₂— or—CH₂CH₂(OCH₂CH₂)_(n)O[CH₂CHO((CH₂CH₂O)_(n)CH₂CH₂)CH₂O]₆(CH₂CH₂O)_(n)CH₂CH₂— or R(O(CH₂CH₂O)_(n)—)₈ orR(O(CH₂CH₂O)_(n)CH₂—)₈ or R(O(CH₂CH₂O)_(n)CH₂CH₂—)₈, in which R includesa tripentaerythritol core), or a derivatized PEG group (e.g., methylether PEG (mPEG), a propylene glycol group, etc.); including dendrimersthereof, copolymers thereof (e.g., having at least two monomers that aredifferent), branched forms thereof, start forms thereof, comb formsthereof, etc., in which n is any useful number in any of these (e.g.,any useful n to provide any useful number average molar mass M_(n)). Yetother linking groups can include a nucleic acid, a peptide, as well asmodified forms thereof.

Exemplary linking agents can include a poly(ethylene glycol) group(e.g., a multivalent poly(ethylene glycol) precursor having a reactivefunctional group, such as an amino group, an ester group, an acrylategroup, a hydroxyl group, a carboxylic acid group, etc.), such as eightarm-PEG amine (8-arm PEG-NH₂, e.g., catalog nos. PSB-811, PSB-812, orPSB-814 available from Creative PEGWorks, Chapel Hill, NC) or aneight-arm PEG succinimidyl ester (such as 8-arm PEG succinimidyl NHSester or 8-arm PEG-SCM (succinimidyl carboxyl methyl ester), e.g.,catalog nos. PSB-841, PSB-842, or PSB-844 available from CreativePEGWorks) or an eight-arm PEG vinylsulfone or an eight-arm PEG hydroxylor a linear PEG thiol or a linear PEG hydroxyl or poly(ethylene glycoldiacrylate) (PEG-DA) or triethylene glycol acrylate (TEGA) or2-carboxyethyl acrylate (CEA) or 2-hydroxyethylacrylate (HEA), as wellas copolymers thereof and/or combinations thereof; an amino acid (e.g.,a poly(amino acid) precursor or a protein, such as a poly(lysine)precursor, a poly(arginine) precursor, lysozyme, avidin, or albumin); aglycerol group (e.g., a poly(glycerol) precursor); a vinyl group (e.g.,a poly(vinyl) precursor or a poly(vinyl alcohol) precursor); ahydroxyacid group (e.g., a poly(lactic acid) precursor, a poly(glycolicacid) precursor, or a poly(lactic-co-glycolic acid) precursor); anacrylate group (e.g., a poly(acrylic acid) precursor or apoly(methacrylic acid) precursor); a silyl ether group (e.g., apoly(silyl ether) precursor); an olefin group (e.g., a poly(acetylene)precursor); and/or an aromatic group (e.g., a poly(pyrrole) precursor, apoly(aniline) precursor, or a poly(thiophene) precursor).

Other exemplary, non-limiting linking agents include3-aminopropyltrimethoxysilane (3-APTMS);(R,S)-1-(3,4-(methylenedioxy)-6-nitrophenyl)ethyl chloroformate(MenPOC); 1-(6-nitrobenzo[d][1,3]dioxol-5-yl)ethyl(3-(trimethoxysilyl)propyl)carbamate; phenyltrichlorosilane (PTCS); anepoxysilane; sulfo-NHS-acetate;1-(3-(trimethoxysilyl)propyl)-1H-pyrrole-2,5-dione;3-glycidoxypropyltrimethoxysilane (3-GPTMS);N-(3-(trimethoxysilyl)propyl)-1H-imidazole-1-carboxamide;N-(6-aminohexyl)-1H-imidazole-1-carboxamide; anhydrides;isocyanotopropyltrimethoxysilane (IPTMS); isocyanates; isothiocyanates;and maleimides.

Yet other non-limiting linking agents include a covalent spacer or anon-covalent spacer. In some embodiments: the spacer may comprise aflexible arm, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15carbon atoms. Exemplary spacers include BS3 ([bis(sulfosuccinimidyl)suberate]; BS3 is a homobifunctional N-hydroxysuccinimide ester thattargets accessible primary amines), NHS/EDC (N-hydroxysuccinimide andN-ethyl-′(dimethylaminopropyl)carbodimide; NHS/EDC allows for theconjugation of primary amine groups with carboxyl groups), sulfo-EMCS([N-e-Maleimidocaproic acid]hydrazide; sulfo-EMCS are heterobifunctionalreactive groups (maleimide and NHS-ester) that are reactive towardsulfhydryl and amino groups), hydrazide (most proteins contain exposedcarbohydrates and hydrazide is a useful reagent for linking carboxylgroups to primary amines), and SATA (N-succinimidyl-S-acetylthioacetate;SATA is reactive towards amines and adds protected sulfhydryls groups).Examples of other suitable spacers are succinic acid, Lys, Glu, Asp, adipeptide such as Gly-Lys, an α-helical spacer (e.g., A(EAAAK)_(n)A,where n is 1, 2, 3, 4, or 5), an alkyl chain (e.g., an optionallysubstituted C₁₋₁₂ alkylene or alkynyl chain), or a polyethylene glycol(e.g., (CH₂CH₂O)_(m), where m is from 1 to 50).

Protecting groups can be employed to protect a reactive group and/or toprovide reduced reactivity (e.g., binding) of an agent (e.g., a captureprobe). Exemplary protecting groups include any described herein,including optionally substituted aryl groups, a poly(ethylene glycol)group, UV-labile groups, etc.).

Functional groups can be present on a spacer, a core, or a cargo. Inaddition, a functional group can include any useful chemical group, suchas a reactive group or a protecting group. In some instances, thelinking agent reacts with a functional group (e.g., present on the cargoor the core), thereby forming an attached spacer that can be furtherreacted with another functional group.

Cargos

The construct can include CRISPR components, as well as other cargos(e.g., associated with the nanoparticle core, with a pore (e.g., by wayof a spacer), and/or within the outer layer). Cargos can include avariety of molecules, including peptides, proteins (e.g., includingprotein complexes, such as a ribonucleoprotein (RNP) complex including anucleic acid and a protein), nucleic acids (e.g., a plasmid, mRNA),aptamers, including antisense oligonucleotides, antibodies, smallmolecule drugs, such as antimicrobials and/or antivirals, alpha/flaviinhibitors, coronavirus (CoV) inhibitors, carbohydrates, dyes, markers,or any other agent described herein.

The cargo can be characterized by a dimension (e.g., a cargo dimension).Exemplary dimensions for the cargo include cargo circumference, cargodiameter, cargo length, and cargo width. Exemplary dimensions (e.g.,cargo circumference, diameter, length, or width) are about 2 nm to about5000 nm (e.g., from 2 nm to 500 nm, 2 nm to 1000 nm, 2 nm to 2500 nm, 5nm to 500 nm, 5 nm to 1000 nm, 5 nm to 2500 nm, 5 nm to 5000 nm, 25 nmto 500 nm, 25 nm to 1000 nm, 25 nm to 2500 nm, 25 nm to 5000 nm, 50 nmto 500 nm, 50 nm to 1000 nm, 50 nm tvary byo 2500 nm, 50 nm to 5000 nm,75 nm to 500 nm, 75 nm to 1000 nm, 75 nm to 2500 nm, 75 nm to 5000 nm,100 nm to 500 nm, 100 nm to 1000 nm, 100 nm to 2500 nm, 100 nm to 5000nm, 500 nm to 1000 nm, 500 nm to 2500 nm, 500 nm to 5000 nm, 750 nm to1000 nm, 750 nm to 2500 nm, 750 nm to 5000 nm, 1000 nm to 2500 nm, 1000nm to 5000 nm, 2500 nm to 5000 nm, or 4000 nm to 5000 nm). Cargo sizecan be determined by dynamic light scattering (DLS) by methods disclosedabove.

Exemplary cargos include an acidic, basic, and hydrophobic drug (e.g.,antiviral agents, antibiotic agents, etc.); a protein (e.g., antibodies,carbohydrates, etc.); a nucleic acid (e.g., DNA, RNA, small interferingRNA (siRNA), minicircle DNA (mcDNA), small hairpin RNA (shRNA),complementary DNA (cDNA), naked DNA, and plasmid, as well as chimeras,single-stranded forms, duplex forms, and multiplex forms thereof andincluding nucleic acid sequences encoding any of these and including oneor more modified nucleic acids); a CRISPR component (e.g., any describedherein, including a guiding component (e.g., any described herein), anuclease, a plasmid, a plasmid that encodes a CRISPR component, aribonucleoprotein complex, a Cas enzyme or an ortholog or homologthereof, a guide RNA, as well as a nucleic acid sequence encoding any ofthese or a complement thereof); a diagnostic/contrast agent, likequantum dots, iron oxide nanoparticles, gadolinium, and indium-111; asmall molecule; a carbohydrate; a drug, a pro-drug, a vitamin, anantibody, a protein, a hormone, a growth factor, a cytokine, a steroid,an anticancer agent, a fungicide, an antimicrobial, an antibiotic, anantiviral agent, etc.; a morphogen; a toxin, e.g., a bacterial proteintoxin; a peptide, e.g., an antimicrobial peptide; an antigen; anantibody; a detection agent (e.g., a particle, such as a conductiveparticle, a microparticle, a nanoparticle, a quantum dot, a latex bead,a colloidal particle, a magnetic particle, a fluorescent particle, etc.;or a dye, such as a fluorescent dye, a luminescent dye, achemiluminescent dye, a colorimetric dye, a radioactive agent, anelectroactive detection agent, etc.); a label (e.g., a quantum dot, ananoparticle, a microparticle, a barcode, a fluorescent label, acolorimetric label, a radio label (e.g., an RF label or barcode),avidin, biotin, a tag, a dye, a marker, an electroactive label, anelectrocatalytic label, and/or an enzyme that can optionally include oneor more linking agents and/or one or more dyes); a capture agent (e.g.,such as a protein that binds to or detects one or more markers (e.g., anantibody or an enzyme), a globulin protein (e.g., bovine serum albumin),a nanoparticle, a microparticle, a sandwich assay reagent, a catalyst(e.g., that reacts with one or more markers), and/or an enzyme (e.g.,that reacts with one or more markers, such as any described herein)); aswell as combinations thereof.

The nucleic acid can be provided in any useful form, such as RNA, DNA,DNA/RNA hybrids, phage, plasmid, linear forms thereof, concatenatedforms thereof, circularized forms thereof, modified forms thereof,single stranded forms thereof, double stranded forms thereof,complements thereof, and encoded forms thereof.

In some instances, the cargo includes a plasmid. The plasmid can encodeany useful CRISPR component (e.g., a guiding component or a nuclease).In addition, the plasmid can express any useful polypeptide and/ornucleic acid sequence, including a nuclear localization sequence, a cellpenetrating peptide, a targeting peptide, a polypeptide toxin, a smallhairpin RNA (shRNA), a small interfering RNA (siRNA), a reporter (e.g.,a reporter protein), etc. Additional reporters include polypeptidereporters which may be expressed by plasmids (such as histone-packagedsupercoiled DNA plasmids) and include polypeptide reporters such asfluorescent green protein and fluorescent red protein. Reporterspursuant to the disclosed technology are utilized principally indiagnostic applications including diagnosing the existence orprogression of a disease state (e.g., diseased tissue) in a subject orpatient and/or the progress of therapy in a patient or subject. Theplasmid can be of any useful form (e.g., supercoiled and/or packagedplasmid). For instance, the plasmid can be a histone-packagedsupercoiled plasmid including a mixture of histone proteins. AdditionalCRISPR components are described herein.

Exemplary anticancer agents include chemotherapeutic agents, such as anagent selected from the group consisting of microtubule-stabilizingagents, microtubule-disruptor agents, alkylating agents,antimetabolites, epidophyllotoxins, antineoplastic enzymes,topoisomerase inhibitors, inhibitors of cell cycle progression, andplatinum coordination complexes, as well as functionalized or modifiedforms thereof (e.g., including polyethylene glycol (PEG)). These may beselected from the group consisting of everolimus, trabectedin, abraxane,TLK 286, AV-299, DN-101, pazopanib, GSK690693, RTA 744, ON 0910.Na, AZD6244 (ARRY-142886), AMN-107, TKI-258, GSK461364, AZD 1152, enzastaurin,vandetanib, ARQ-197, MK-0457, MLN8054, PHA-739358, R-763, AT-9263, aFLT-3 inhibitor, a VEGFR inhibitor, an EGFR TK inhibitor, an aurorakinase inhibitor, a PIK-1 modulator, a Bcl-2 inhibitor, an HDACinhibitor, a c-MET inhibitor, a PARP inhibitor, a Cdk inhibitor, an EGFRTK inhibitor, an IGFR-TK inhibitor, an anti-HGF antibody, a PI3 kinaseinhibitors, an AKT inhibitor, a JAK/STAT inhibitor, a checkpoint-1 or 2inhibitor, a focal adhesion kinase inhibitor, a Map kinase kinase (mek)inhibitor, a VEGF trap antibody, pemetrexed, erlotinib, dasatanib,nilotinib, decatanib, panitumumab, amrubicin, oregovomab, Lep-etu,nolatrexed, azd2171, batabulin, ofatumumab, zanolimumab, edotecarin,tetrandrine, rubitecan, tesmilifene, oblimersen, ticilimumab,ipilimumab, gossypol, Bio 111, 131-I-TM-601, ALT-110, BIO 140, CC 8490,cilengitide, gimatecan, IL13-PE38QQR, INO 1001, IPdR₁ KRX-0402,lucanthone, LY 317615, neuradiab, vitespan, Rta 744, Sdx 102,talampanel, atrasentan, XR 311, romidepsin, ADS-100380, sunitinib,5-fluorouracil, vorinostat, etoposide, etoposide phosphate, gemcitabine,doxorubicin, liposomal doxorubicin, 5′-deoxy-5-fluorouridine,vincristine, temozolomide, ZK-304709, seliciclib, PD0325901, AZD-6244,capecitabine, L-glutamic acid, N-[4-[2-(2-amino-4,7-dihydro-4-oxo-1H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoyl]-, disodium salt,heptahydrate, camptothecin, PEG-labeled irinotecan, tamoxifen,toremifene citrate, anastrazole, exemestane, letrozole,DES(diethylstilbestrol), estradiol, estrogen, conjugated estrogen,bevacizumab, IMC-1C11, CHIR-258,3-[5-(methylsulfonylpiperadinemethyl)-indolyl]-quinolone, vatalanib,AG-013736, AVE-0005, the acetate salt of [D-Ser(But)6,Azgly 10](pyro-Glu-His-Trp-Ser-Tyr-D-Ser(But)-Leu-Arg-Pro-Azgly-NH₂ acetate[C₅₉H₈₄N₁₈O₁₄—(C₂H₄O₂)_(X) where x=1 to 2.4], goserelin acetate,leuprolide acetate, triptorelin pamoate, medroxyprogesterone acetate,hydroxyprogesterone caproate, megestrol acetate, raloxifene,bicalutamide, flutamide, nilutamide, megestrol acetate, CP-724714,TAK-165, HKI-272, erlotinib, lapatanib, canertinib, ABX-EGF antibody,erbitux, EKB-569, PKI-166, GW-572016, lonafarnib, BMS-214662,tipifarnib, amifostine, NVP-LAQ824, suberoyl analide hydroxamic acid,valproic acid, trichostatin A, FK-228, SU11248, sorafenib, KRN951,adriamycin, aminoglutethimide, arnsacrine, anagrelide, L-asparaginase,Bacillus Calmette-Guerin (BCG) vaccine, bleomycin, buserelin, busulfan,carboplatin, carmustine, chlorambucil, cisplatin, cladribine,clodronate, cyproterone, cytarabine, dacarbazine, dactinomycin,daunorubicin, diethylstilbestrol, epirubicin, fludarabine,fludrocortisone, fluoxymesterone, flutamide, gemcitabine, hydroxyurea,idarubicin, ifosfamide, imatinib (e.g., including imatinib mesylate),leuprolide, levamisole, lomustine, mechlorethamine, melphalan,6-mercaptopurine, mesna, methotrexate, mitomycin, mitotane,mitoxantrone, nilutamide, octreotide, oxaliplatin, pamidronate,pentostatin, plicamycin, porfimer, procarbazine, raltitrexed, rituximab,streptozocin, teniposide, testosterone, thalidomide, thioguanine,thiotepa, tretinoin, vindesine, 13-cis-retinoic acid, phenylalaninemustard, uracil mustard, estramustine, altretamine, floxuridine,5-deooxyuridine, cytosine arabinoside, 6-mecaptopurine, deoxycoformycin,calcitriol, valrubicin, mithramycin, vinblastine, vinorelbine,topotecan, razoxin, marimastat, COL-3, neovastat, BMS-275291,squalamine, endostatin, SU5416, SU6668, EMD121974, interleukin-12,IM862, angiostatin, vitaxin, droloxifene, idoxyfene, spironolactone,finasteride, cimitidine, trastuzumab, denileukin diftitox, gefitinib,bortezimib, paclitaxel, cremophor-free paclitaxel, docetaxel, epithiloneB, BMS-247550, BMS-310705, droloxifene, 4-hydroxytamoxifen,pipendoxifene, ERA-923, arzoxifene, fulvestrant, acolbifene,lasofoxifene, idoxifene, TSE-424, HMR-3339, ZK186619, topotecan,PTK787/ZK 222584, VX-745, PD 184352, rapamycin,40-O-(2-hydroxyethyl)-rapamycin, temsirolimus, AP-23573, RAD001,ABT-578, BC-210, LY294002, LY292223, LY292696, LY293684, LY293646,wortmannin, ZM336372, L-779,450, PEG-filgrastim, darbepoetin,erythropoietin, granulocyte colony-stimulating factor, zolendronate,prednisone, cetuximab, granulocyte macrophage colony-stimulating factor,histrelin, pegylated interferon alfa-2a, interferon alfa-2a, pegylatedinterferon alfa-2b, interferon alfa-2b, azacitidine, PEG-L-asparaginase,lenalidomide, gemtuzumab, hydrocortisone, interleukin-1, dexrazoxane,alemtuzumab, all-transretinoic acid, ketoconazole, interleukin-2,megestrol, immune globulin, nitrogen mustard, methylprednisolone,ibritgumomab tiuxetan, androgens, decitabine, hexamethylmelamine,bexarotene, tositumomab, arsenic trioxide, cortisone, editronate,mitotane, cyclosporine, liposomal daunorubicin, Edwina-asparaginase,strontium 89, casopitant, netupitant, an NK-1 receptor antagonists,palonosetron, aprepitant, diphenhydramine, hydroxyzine, metoclopramide,lorazepam, alprazolam, haloperidol, droperidol, dronabinol,dexamethasone, methylprednisolone, prochlorperazine, granisetron,ondansetron, dolasetron, tropisetron, pegfilgrastim, erythropoietin,epoetin alfa, and darbepoetin alfa, among others. In some embodiments,the anticancer agent is selected from the group of doxorubicin,melphalan, bevacizumab, dactinomycin, cyclophosphamide, doxorubicinliposomal, amifostine, etoposide, gemcitabine, altretamine, topotecan,cyclophosphamide, paclitaxel, carboplatin, cisplatin, and taxol.

Exemplary antiviral agents (e.g., anti-HV agents) include, for example,nucleoside reverse transcriptase inhibitors (NRTI), non-nucleosidereverse transcriptase inhibitors (NNRTI), protease inhibitors, fusioninhibitors, among others, exemplary compounds of which may include, forexample, 3TC (Lamivudine), AZT (Zidovudine), (−)-FTC, ddI (Didanosine),ddC (zalcitabine), abacavir (ABC), tenofovir (PMPA), D-D4FC (Reverset),D4T (Stavudine), Racivir, L-FddC, L-FD4C, NVP (Nevirapine), DLV(Delavirdine), EFV (Efavirenz), SQVM (Saquinavir mesylate), RTV(Ritonavir), IDV (Indinavir), SQV (Saquinavir), NFV (Nelfinavir), APV(Amprenavir), LPV (Lopinavir), fusion inhibitors such as T20, amongothers, fuseon and mixtures thereof, including anti-HIV compoundspresently in clinical trials or in development. Exemplary anti-HBVagents include, for example, hepsera (adefovir dipivoxil), lamivudine,entecavir, telbivudine, tenofovir, emtricitabine, clevudine,valtoricitabine, anidoxovir, pradefovir, racivir, BAM 205, nitazoxanide,UT 231-B, Bay 41-4109, EHT899, zadaxin (thymosin alpha-1) and mixturesthereof. Anti-HCV agents include, for example, interferon pegylatedintergeron, ribavirin, NM 283, VX-950 (telaprevir), SCH 50304, TMC435,VX-500, BX-813, SCH503034, R1626, ITMN-191 (R7227), R7128, PF-868554,TT033, CGH-759, GI 5005, MK-7009, SIRNA-034, MK-0608, A-837093, GS 9190,ACH-1095, GSK625433, TG4040 (MVA-HCV), A-831, F351, NS5A, NS4B, ANA598,A-689, GNI-104, IDX102, ADX184, GL59728, GL60667, PSI-7851, TLR9Agonist, PHX1766, SP-30 and mixtures thereof.

Other exemplary antiviral agents include broad spectrum antiviralagents, antibodies, small molecule antiviral agents, antiretroviralagents, etc. Further non-limiting antiviral agents include abacavir,ACH-3102, acyclovir (acyclovir), acyclovir, adefovir, amantadine,amprenavir, ampligen, arbidol, asunaprevir, atazanavir, atripla,balavir, BCX4430, boceprevir, brincidofovir, brivudine, cidofovir,clevudine, combivir, cytarabine, daclatasvir, dasabuvir, deleobuvir,dolutegravir, darunavir, delavirdine, didanosine, docosanol, edoxudine,efavirenz, elbasvir, emtricitabine, enfuvirtide, entecavir, ecoliever,faldaprevir, famciclovir, favipiravir, fomivirsen, fosamprenavir,foscarnet, fosfonet, ganciclovir, grazoprevir, ibacitabine, imunovir,idoxuridine, imiquimod, indinavir, interferon type III, interferon typeII, interferon type I, interferon, interferon alfa 2b, lamivudine,laninamivir, ledipasvir (with or without sofosbuvir), lopinavir,loviride, maraviroc, moroxydine, methisazone, MK-3682, MK-8408,nelfinavir, nevirapine, nexavir, novir, ombitasvir (with or withoutparitaprevir and/or ritonavir), oseltamivir (Tamiflu), paritaprevir,peginterferon alfa-2a, penciclovir, peramivir, pleconaril,podophyllotoxin, raltegravir, resiquimod, ribavirin, rifampicin,rimantadine, ritonavir, pyramidine, samatasvir, saquinavir, simeprevir,sofosbuvir, stavudine, taribavirin, tecovirimat (ST-246), telaprevir,telbivudine, tenofovir, tenofovir disoproxil, tipiracil, tipranavir,trifluridine (with or without tipiracil), trizivir, tromantadine,truvada, umifenovir, valaciclovir (Valtrex), valganciclovir, vicriviroc,vidarabine, viramidine, zalcitabine, zanamivir (Relenza), zidovudine,including prodrugs, salts, and/or combinations thereof.

Exemplary antibiotics or antibacterial agents include gentamicin,kanamycin, neomycin, netilmicin, tobramycin, paromomycin, spectinomycin,geldanamycin, herbimycin, rifaximin, streptomycin, ertapenem, doripenem,imipenem/cilastatin, meropenem, cefadroxil, cefazolin, cephalothin,cephalexin, cefaclor, cefamandole, cefoxitin, cefprozil, cefuroxime,cefixime, cefdinir, cefditoren, cefoperazone cefotaxime, cefpodoxime,ceftazadime, ceftibuten, ceftizoxime ceftriaxone, cefepime, ceftarolinefosamil, ceftobiprole, teicoplanin, vancomycin, telavancin, daptomycin,oritavancin, WAP-8294A, azithromycin, clarithromycin, dirithromycin,erythromycin, roxithromycin, telithromycin, spiramycin, clindamycin,lincomycin, aztreonam, furazolidone, nitrofurantoin, oxazolidonones,linezolid, posizolid, radezolid, torezolid, amoxicillin, ampicillin,azlocillin, carbenicillin, cloxacillin dicloxacillin, flucloxacillin,mezlocillin, methicillin, nafcillin, oxacillin, penicillin G, penicillinV, piperacillin, temocillin, ticarcillin, amoxicillin/clavulanate,ampicillin/sulbactam, piperacillin/tazobactam, ticarcillin/clavulanate,bacitracin, colistin, polymyxin B, ciprofloxacin, enoxacin,gatifloxacin, gemifloxacin, levofloxacin, lomefloxacin, moxifloxacin,nalidixic acid, norfloxacin, ofloxacin, trovafloxacin, grepafloxacin,sparfloxacin, mafenide, sulfacetamide, sulfadiazine, sulfadimethoxine,sulfamethizole, sulfamethoxazole, sulfasalazine, sulfisoxazole,trimethoprim-sulfamethoxazole, sulfonamidochrysoidine, demeclocycline,doxycycline, vibramycin minocycline, tigecycline, oxytetracycline,tetracycline, clofazimine, capreomycin, cycloserine, ethambutol,rifampicin, rifabutin, rifapentine, arsphenamine, chloramphenicol,fosfomycin, fusidic acid, metronidazole, mupirocin, platensimycin,quinupristin/dalfopristin, thiamphenicol, tigecycline, and tinidazoleand combinations thereof.

CRISPR Components

In an embodiment, the cargo includes a CRISPR component. CRISPRcomponent includes any employing a nucleic acid sequence capable ofrecruiting a CRISPR-associated (Cas) protein to achieve geneticmodification. An exemplary CRISPR component includes those having atrans-acting CRISPR RNA (tracrRNA) and CRISPR RNA (crRNA) fused into asingle, synthetic ‘guide RNA’ that directs a Cas nuclease (e.g., Cas9)to virtually any desired DNA sequence (see, e.g., FIG. 6 ). Thesynthetic guide RNA (gRNA) can include at least three differentportions: a first portion including the tracrRNA sequence, a secondportion including the crRNA sequence, and a third portion including atargeting portion or a genomic specific sequence (gsRNA) that binds to adesired genomic target sequence (e.g., genomic target DNA sequence,including a portion or a strand thereof). The chimeric tracrRNA-crRNAsequence facilitates binding and recruitment of the endonuclease (e.g.,Cas9), and the gsRNA sequence provides site-specificity to the targetnucleic acid, thereby allowing Cas9 to selectively introducesite-specific breaks in the target.

In any embodiment herein, the cargo can include a CRISPR component.Exemplary CRISPR components can include a guide RNA, a Cas enzyme, and anucleic acid sequence (e.g., a plasmid) encoding any of these. Yet otherexemplary CRISPR components are shown in FIGS. 12, 13A-13C, 14A-14H, 15,16A-16C, 17, 18, and 19 , including, as applicable, a nucleic acidsequence encoding any of these (e.g., a nucleic acid sequence encodingany polypeptide sequence therein, such as SEQ ID Nos: 110-117 or afragment thereof), a polypeptide generated by any nucleic acid sequencetherein, as well as a complement of any nucleic acid sequence therein(e.g., a nucleic acid sequence that is a complement of any one of SEQ IDNos: 20-32, 40-54, 60-65, 80-93, 100-103, or a fragment thereof).

In particular embodiments, the particle can include one or more CRISPRcomponents (e.g., associated with or within a pore of the core (e.g., byway of a spacer), associated with a surface of the core, and/or withinthe outer layer).

FIG. 6 and FIG. 7A-7C show exemplary CRISPR components. FIG. 6 shows anexemplary CRISPR component that includes a guiding component 90 to bindto the target sequence 97, as well as a nuclease 98 (e.g., a Casnuclease or an endonuclease, such as a Cas endonuclease) that interactswith the guiding component and the target sequence.

FIG. 7(A) shows a non-limiting guiding component 300 having a targetingportion 304, a first portion 301, a second portion 302, and a linker 303disposed between the first and second portions. FIG. 7(B) shows anothernon-limiting guiding component 350 having a targeting portion 354, afirst portion 351, a second portion 352 having a hairpin, and a linker353 disposed between the first and second portions. Fig. (C) showsnon-limiting interactions between the guiding component 400, the genomicsequence 412, and the first and second portion 401,402. As can be seen,the target sequence 411 of the genomic sequence 412 is targeted by wayof non-covalent binding 421 to the targeting portion 404, and secondarystructure can be optionally implemented by way of non-covalent binding422 between the first portion 401 and the second portion 402. Thetargeting portion 404, first portion 401, linker 403, and second portion402 can be attached in any useful manner (e.g., to provide a 5′ end 405and a 3′ end 406).

This CRISPR/Cas system can be adapted to control genetic expression intargeted manner, such as, e.g., by employing synthetic, non-naturallyoccurring constructs that use crRNA nucleic acid sequences, tracrRNAnucleic acid sequences, and/or Cas polypeptide sequences, as well asmodified forms thereof.

In an embodiment, a CRISPR component includes a guiding component. Ingeneral, the guiding component includes a nucleic acid sequence (e.g., asingle polynucleotide) that includes at least two portions: a targetingportion, which is a nucleic acid sequence that imparts specifictargeting to the target genomic locus (e.g., a guide RNA or gRNA); andan interacting portion, which is another nucleic acid sequence thatbinds to a nuclease (e.g., a Cas endonuclease). In some instances, theinteracting portion includes two particular sequences that bind thenuclease, e.g., a short crRNA sequence attached to the guide nucleicacid sequence; and a tracrRNA sequence attached to the crRNA sequence.Exemplary targeting CRISPR components include a minicircle DNA vectoroptimized for in vivo expression.

Another CRISPR component includes a nuclease (e.g., that binds thetargeting nucleic acid sequence). The nuclease CRISPR component caneither be an enzyme, or a nucleic acid sequence that encodes for thatenzyme. Exogenous endonuclease (e.g., Cas9) can be encoded by a cargostored within the construct. In an embodiment, a nuclease such as Cas9(e.g., SEQ ID NO:110) is employed, as well as nickase forms anddeactivated forms (e.g., SEQ ID NO:111) thereof (e.g., including one ormore mutations, such as D10A, H840A, N854A, and N863A in SEQ ID NO:110or in an amino acid sequence sufficiently aligned with SEQ ID NO:110),including nucleic acid sequences that encode for such nuclease.Pathogen-directed and host-directed CRISPR components (e.g., guidingcomponents and/or nuclease), as well as minicircle DNA vectors thatencode Cas and guiding components can be developed. The nuclease can beconfigured to bind the target sequence and/or cleave the targetsequence.

Non-limiting examples of nucleases are described in FIG. 8A-8H. In someembodiments, a vector comprises a regulatory element operably linked toan enzyme-coding sequence encoding a nuclease (e.g., a CRISPR enzyme,such as a Cas protein). Non-limiting examples of Cas proteins includeCas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also knownas Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2,Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6,Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15,Csf1, Csf2, Csf3, Csf4, homologs thereof, or modified versions thereof.These enzymes are known; for example, the amino acid sequence of S.pyogenes Cas9 protein may be found in the SwissProt database underaccession number Q99ZW2. In some embodiments, the unmodified CRISPRenzyme has DNA cleavage activity, such as Cas9. In some embodiments theCRISPR enzyme is Cas9, and may be Cas9 from S. pyogenes or S.pneumoniae. In some embodiments, the CRISPR enzyme directs cleavage ofone or both strands at the location of a target sequence, such as withinthe target sequence and/or within the complement of the target sequence.In some embodiments, the CRISPR enzyme directs cleavage of one or bothstrands within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100,200, 500, or more base pairs from the first or last nucleotide of atarget sequence.

The nuclease may be a Cas9 homolog or ortholog. In some embodiments, thenuclease is codon-optimized for expression in a eukaryotic cell. In someembodiments, the nuclease directs cleavage of one or two strands at thelocation of the target sequence. In some embodiments, the nuclease lacksDNA strand cleavage activity. In some embodiments, the first regulatoryelement is a polymerase III promoter. In some embodiments, the secondregulatory element is a polymerase II promoter.

Any useful Cas protein or complex can be employed. Exemplary Casproteins or complexes include those involved in Type I, Type II, or TypeIII CRISPR/Cas systems, including but not limited to theCRISPR-associated complex for antiviral defense (Cascade, including aRAMP protein), Cas3 and/or Cas 7 (e.g., for Type I systems, such as TypeI-E systems), Cas9 (formerly known as Csn1 or Csx12, e.g., such as inType II systems), Csm (e.g., in Type III-A systems), Cmr (e.g., in TypeIII-B systems), Cas10 (e.g., in Type III systems), as well assubassemblies or sub-components thereof and assemblies including suchCas proteins or complexes. Additional Cas proteins and complexes aredescribed in Makarova K S et al., “Evolution and classification of theCRISPR-Cas systems,” Nat. Rev. Microbiol. 2011; 9:467-77, which isincorporated herein by reference in its entirety.

In some embodiments, a vector encodes a CRISPR enzyme that is mutatedwith respect to a corresponding wild-type enzyme such that the mutatedCRISPR enzyme lacks the ability to cleave one or both strands of atarget polynucleotide containing a target sequence. For example, anaspartate-to-alanine substitution (D10A) in the RuvC I catalytic domainof Cas9 from S. pyogenes converts Cas9 from a nuclease that cleaves bothstrands to a nickase (cleaves a single strand). Other examples ofmutations that render Cas9 a nickase include, without limitation, H840A,N854A, and N863A. In aspects of the disclosed technology, nickases maybe used for genome editing via homologous recombination. In someinstances, the Cas protein includes a modification of one of more ofD10A, H840A, N854A, and N863A in SEQ ID NO:110 or in an amino acidsequence sufficiently aligned with SEQ ID NO:110.

As a further example, two or more catalytic domains of Cas9 (RuvC I,RuvC II, and RuvC III) may be mutated to produce a mutated Cas9substantially lacking all DNA cleavage activity. In some embodiments, aD10A mutation is combined with one or more of H840A, N854A, or N863Amutations to produce a Cas9 enzyme substantially lacking all DNAcleavage activity. In some embodiments, a CRISPR enzyme is considered tosubstantially lack all DNA cleavage activity when the DNA cleavageactivity of the mutated enzyme is less than about 25%, 10%, 5%, 1%,0.1%, 0.01%, or lower with respect to its non-mutated form. Othermutations may be useful; where the Cas9 or other CRISPR enzyme is from aspecies other than S. pyogenes, mutations in corresponding amino acidsmay be made to achieve similar effects.

In some embodiments, the guiding component comprises a modification orsequence that provides for an additional desirable feature (e.g.,modified or regulated stability; subcellular targeting; tracking, e.g.,a fluorescent label; a binding site for a protein or protein complex;etc.). Non-limiting examples include: a short motif (referred to as theprotospacer adjacent motif (PAM)); a 5′ cap (e.g., a 7-methylguanylatecap (m7G)); a 3′ polyadenylated tail (i.e., a 3′ poly(A) tail); ariboswitch sequence (e.g., to allow for regulated stability and/orregulated accessibility by proteins and/or protein complexes); astability control sequence; a sequence that forms a dsRNA duplex (i.e.,a hairpin)); a modification or sequence that targets the RNA to asubcellular location (e.g., nucleus, mitochondria, and chloroplasts); amodification or sequence that provides for tracking (e.g., directconjugation to a fluorescent molecule, conjugation to a moiety thatfacilitates fluorescent detection, a sequence that allows forfluorescent detection, etc.); a modification or sequence that provides abinding site for proteins (e.g., proteins that act on DNA, includingtranscriptional activators, transcriptional repressors, DNAmethyltransferases, DNA demethylases, histone acetyltransferases, andhistone deacetylases); and combinations thereof.

A guiding component and a nuclease can form a complex (i.e., bind vianon-covalent interactions). The guiding component provides targetspecificity to the complex by comprising a nucleotide sequence that iscomplementary to a sequence of a target sequence. The nuclease of thecomplex provides the site-specific activity. In other words, thenuclease is guided to a target sequence (e.g., a target sequence in achromosomal nucleic acid; a target sequence in an extrachromosomalnucleic acid, e.g., an episomal nucleic acid, a minicircle, etc.; atarget sequence in a mitochondrial nucleic acid; a target sequence in achloroplast nucleic acid; a target sequence in a plasmid; etc.) byvirtue of its association with the protein-binding segment (e.g., theinteracting portion) of the guiding component.

In some embodiments, the guiding component comprises two separatenucleic acid molecules (e.g., a separate targeting portion and aseparate interacting portion; a separate first portion and a separatesecond portion; or a separate targeting portion-first portion that iscovalently bound and a separate second portion). In other embodiments,the guiding component is a single nucleic acid molecule including acovalent bond or a linker between each separate portion (e.g., atargeting portion covalently linked to an interacting portion).

FIG. 6 shows an exemplary CRISPR component that includes a guidingcomponent 90 to bind to the target sequence 97, as well as a nuclease 98(e.g., a Cas nuclease or an endonuclease, such as a Cas endonuclease)that interacts with the guiding component and the target sequence. Ascan be seen, the guiding component 90 includes a targeting portion 94configured to bind to the target sequence 97 of a genomic sequence 96(e.g., a target sequence having substantially complementarity with thegenomic sequence or a portion thereof). In this manner, the targetingportion confers specificity to the guiding component, thereby allowingcertain target sequences to be activated, inactivated, and/or modified.

The guiding component 90 also includes an interacting portion 95, whichin turn is composed of a first portion 91, a second portion 92, and alinker 93 that covalently links the first and second portions. Theinteracting portion 95 is configured to recruit the nuclease (e.g., aCas nuclease) in proximity to the site of the target sequence. Thus, theinteracting portion includes nucleic acid sequences that providepreferential binding (e.g., specific binding) of the nuclease. Once inproximity, the nuclease 98 can bind and/or cleave the target sequence ora sequence in proximity to the target sequence in a site-specificmanner.

The first portion, second portion, and linker can be derived in anyuseful manner. In one instance, the first portion can include a crRNAsequence, a consensus sequence derived from known crRNA sequences, amodified crRNA sequence, or an entirely synthetic sequence known to binda Cas nuclease or determined to competitively bind a Cas nuclease whencompared to a known crRNA sequence. Exemplary sequences for a firstportion are described in FIG. 9 (SEQ ID NOs:20-32). Another exemplarysequence for a first portion is 5′-GUUUUAGAGCUA-3′ (SEQ ID NO:70). Insome embodiments, the first portion is a nucleic acid sequence having atleast 80% sequence identity (e.g., at least 85%, 90%, 95%, or 99%sequence identity) to any one of SEQ ID NOs:20-32 and 70 or a complementof any of these, or a fragment thereof (e.g., having a length of about4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24,26, 28, 30, 32, 34, 36, 38, 40, or more nucleotides).

In some embodiments, the first portion is a crRNA sequence that exhibitsat least 50%, 60%, 70%, 80%, 90%, 95%, or 99% of sequencecomplementarity to any one of SEQ ID NOs:20-32 and 70. In otherembodiments, the first portion is a fragment (e.g., having a length ofabout 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22,24, 26, 28, 30, 32, 34, 36, 38, 40, or more nucleotides) of a crRNAsequence that exhibits at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% ofsequence complementarity to any one of SEQ ID NOs:20-32 and 70.

In another instance, the second portion can include a tracrRNA sequence,a consensus sequence derived from known tracrRNA sequences, a modifiedtracrRNA sequence, or an entirely synthetic sequence known to bind a Casnuclease or determined to competitively bind a Cas nuclease whencompared to a known tracrRNA sequence. Exemplary sequences for a secondportion are described in FIG. 10A-10C (SEQ ID NOs:40-54) and in FIG. 11(SEQ ID NOs:60-65). Another exemplary sequence for a second portion is5′-UAGCAAGUUAAAA UAAGGCUAGUCCG-3′ (SEQ ID NO:71).

In some embodiments, the second portion is a nucleic acid sequencehaving at least 80% sequence identity (e.g., at least 85%, 90%, 95%, or99% sequence identity) to any one of SEQ ID NOs:40-54, 60-65, and 71 ora complement of any of these, or a fragment thereof (e.g., having alength of about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, or more nucleotides).

In some embodiments, the second portion is a tracrRNA sequence thatexhibits at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% of sequencecomplementarity to any one of SEQ ID NOs:40-54, 60-65, and 71. In otherembodiments, the second portion is a fragment (e.g., having a length ofabout 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22,24, 26, 28, 30, 32, 34, 36, 38, 40, or more nucleotides) of a tracrRNAsequence that exhibits at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% ofsequence complementarity to any one of SEQ ID NOs:40-54, 60-65, and 71.

The linker can be, for example, one or more transcribable elements, suchas a nucleotide or a nucleic acid, or including one or more chemicallinkers. Further, the linker can be derived from a fragment of anyuseful tracrRNA sequence (e.g., any described herein). The first andsecond portions can interact in any useful manner. For example, thefirst portion can have a sequence portion that is sufficientlycomplementary to a sequence portion of the second portion, therebyfacilitating duplex formation or non-covalent bonding between the firstand second portion. In another example, the second portion can include afirst sequence portion that is sufficiently complementary to a secondsequence portion, thereby facilitating hairpin formation within thesecond portion. Further CRISPR components are described in FIG. 7A-7C.

In another embodiment, the guiding component has a structure of A-L-B,in which A includes a first portion (e.g., any one of SEQ ID NOs:20-32and 70, or a fragment thereof), L is a linker (e.g., a covalent bond, anucleic acid sequence, a fragment of any one of SEQ ID NOs:40-54, 60-65,and 71, or any other useful linker or spacer described herein), and B isa second portion (e.g., any one of SEQ ID NOs:40-54, 60-65, and 71, or afragment thereof) (FIG. 12 ). In another embodiment, the guidingcomponent is a sequence having at least 80% sequence identity (e.g., atleast 85%, 90%, 95%, or 99% sequence identity) to any one SEQ IDNOs:80-93, or a fragment thereof.

In yet another embodiment, the guiding component is a sequence thatexhibits at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% of sequencecomplementarity to any one SEQ ID NOs:100-103, or a fragment thereof(FIG. 13 ). In another embodiment, the guiding component is a sequencehaving at least 80% sequence identity (e.g., at least 85%, 90%, 95%, or99% sequence identity) to any one SEQ ID NOs:100-103, or a fragmentthereof.

In some embodiments, the CRISPR component includes ds plasmid DNA, whichis modified to express RNA and/or a protein. In other embodiments, theCRISPR component is supercoiled and/or packaged (e.g., within a complex,such as those containing histones, lipids (e.g., lipoplexes), proteins(e.g., cationic proteins), cationic carrier, nanoparticles (e.g., goldor metal nanoparticles), etc.), which may be optionally modified with anuclear localization sequence (e.g., a peptide sequence incorporated orotherwise crosslinked into histone proteins, which comprise thehistone-packaged supercoiled plasmid DNA). Other exemplary histoneproteins include H1, H2A, H2B, H3 and H4, e.g., in a ratio of 1:2:2:2:2with optional nuclear localization sequences (e.g., any describedherein, such as SEQ ID NOs:9-12).

The CRISPR component can include any useful promoter sequence(s),expression control sequence(s) that controls and regulates thetranscription and translation of another DNA sequence, and signalsequence(s) that encodes a signal peptide. The promoter sequence caninclude a DNA regulatory region capable of binding RNA polymerase in acell and initiating transcription of a downstream (3′ direction) codingsequence. For purposes of the present disclosed technology, the promotersequence is bounded at its 3′ terminus by the transcription initiationsite and extends upstream (5′ direction) to include the minimum numberof bases or elements necessary to initiate transcription at levelsdetectable above background. Within the promoter sequence will be founda transcription initiation, as well as protein binding domains(consensus sequences) responsible for the binding of RNA polymerase.Eukaryotic promoters will often, but not always, contain “TATA” boxesand “CAT” boxes. Prokaryotic promoters contain Shine-Dalgamo sequencesin addition to the −10 and −35 consensus sequences.

In addition, the CRISPR components can be formed from any usefulcombination of one or more nucleic acids (or a polymer of nucleic acids,such as a polynucleotide). Exemplary nucleic acids or polynucleotides ofthe disclosed technology include, but are not limited to, ribonucleicacids (RNAs), deoxyribonucleic acids (DNAs), threose nucleic acids(TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs),locked nucleic acids (LNAs, including LNA having a β-D-riboconfiguration, α-LNA having an α-L-ribo configuration (a diastereomer ofLNA), 2′-amino-LNA having a 2′-amino functionalization, and2′-amino-α-LNA having a 2′-amino functionalization) or hybrids,chimeras, or modified forms thereof. Exemplary modifications include anyuseful modification, such as to the sugar, the nucleobase, or theinternucleoside linkage (e.g., to a linking phosphate/to aphosphodiester linkage/to the phosphodiester backbone). One or moreatoms of a pyrimidine nucleobase may be replaced or substituted withoptionally substituted amino, optionally substituted thiol, optionallysubstituted alkyl (e.g., methyl or ethyl), or halo (e.g., chloro orfluoro). In certain embodiments, modifications (e.g., one or moremodifications) are present in each of the sugar and the internucleosidelinkage. Modifications according to the present disclosure may bemodifications of ribonucleic acids (RNAs) to deoxyribonucleic acids(DNAs), threose nucleic acids (TNAs), glycol nucleic acids (GNAs),peptide nucleic acids (PNAs), locked nucleic acids (LNAs) or hybridsthereof). Additional modifications are described herein.

Toxicity of CRISPR components, to the host, can be minimized. Forinstance, toxicity can result from protocells or carriers due toexpression of Cas9 products or immune responses. Specifically, thelifetime of CRISPR components in the cell can be controlled by addingfeatures that are stabilized or destabilized with cellular proteases, byinducing expression only under a microbial or viral promoter, and byusing guiding components with modified backbones (e.g., 2-OMe) tominimize immune recognition.

Resistance to CRISPR components can be minimized. Any single antibioticor antiviral countermeasure is prone to the development of resistance,so pathogens will likely mutate around individual guiding componenttargets. However, the development of resistance can be prevented bytargeting orthogonal mechanisms via multiplexed guiding components incombination with current antivirals/antimicrobials.

Off-target mutations or genetic modification can be minimized. Forinstance, bioinformatic guiding component design programs can be used todetermine minimal effective CRISPR component doses. If needed, thenickase version of Cas9 can be employed.

The CRISPR component can be employed to target a nucleic acid sequence(e.g., present in the host's genomic sequence and/or the pathogen'sgenomic sequence). In one instance, the target sequence can include asequence present in the host's genomic sequence in order, e.g.,activate, inactive, or modify expression of factor or proteins withinthe host's cellular machinery. For instance, the target sequence canbind to one or more genomic sequences for an immunostimulatory proteinthat, upon expression, would enhance the immune response by the host toan infection. Pathogens are known to down-regulate proteins that wouldotherwise assist in recognizing non-self protein motifs. Thus, inanother instance, the target sequence can bind to one or more regulatorproteins and enhance their transcription and expression. In yet anotherinstance, one or more polypeptides may be up-regulated, as compared tothe normal basal rate, and such up-regulation may be modified by thepresence of the pathogen. Accordingly, the target sequence can beemployed to bind to one or more up-regulated polypeptides in order toinactivate or repress transcription/expression of those polypeptides.

An exemplary target sequence (e.g., in a host or subject) includes,without limitation, a nucleic acid sequence encoding animmunostimulatory protein, a cluster of differentiation protein, a cellsurface protein, a pathogen receptor protein (e.g., a pathogenrecognition receptor, such as TLR9), a glycoprotein (e.g.,granulocyte-colony stimulating factor), a cytokine (e.g., interferon ortransforming growth factor beta (TGF-beta)), a pattern recognitionreceptor protein, a hormone (e.g., a prostaglandin), or a helicaseenzyme.

In yet another instance, the target sequence can be employed toactivate, inhibit, and/or modify a target sequence (e.g., associatedwith the presence of a pathogen, a tumor, etc.). For instance, thetarget sequence can be configured to activate one or more targetsequences encoding proteins that promote programmed cell death orapoptosis (e.g., of the pathogen or of particular tissue types, such asmetastatic growths, tumors, lesions, etc.). For instance, the targetsequence can be configured to inactivate or modify one or more targetsequences encoding proteins that are suppressed by the pathogen.Exemplary target sequence (e.g., in a pathogen) includes, withoutlimitation, a nucleic acid sequence encoding a virulence factor (e.g., alipase, a protease, a nuclease (e.g., a DNAse or an RNase), a hemolysin,a hyaluronidase, an immunoglobulin protease, an endotoxin, or anexotoxin), a cell surface protein (e.g., an adhesion), an envelopeprotein (e.g., a phospholipid, a lipopolysaccharide, a lipoprotein, or apolysaccharide), a glycoprotein, a polysaccharide protein, atransmembrane protein (e.g., an invasin), or a regulatory protein.

The CRISPR component can be employed to activate the target sequence(e.g., the Cas polypeptide can include one or more transcriptionalactivation domains, which upon binding of the Cas polypeptide to thetarget sequence, results in enhanced transcription and/or expression ofthe target sequence), inactivate the target sequence (e.g., the Caspolypeptide can bind to the target sequence, thereby inhibitingexpression of one or more proteins encoded by the target sequence; theCas polypeptide can introduce double-stranded or single-stranded breaksin the target sequence, thereby inactivating the gene; or the Caspolypeptide can include one or more transcriptional repressor domains,which upon binding of the Cas polypeptide to the target sequence,results in reduced transcription and/or expression of the targetsequence), and/or modify the target sequence (e.g., the Cas polypeptidecan cleave the target sequence of the pathogen and optionally inserts afurther nucleic acid sequence).

Any useful transcriptional activation domains can be employed (e.g.,VP64, VP16, HIV TAT, or a p65 subunit of nuclear factor KB). Inparticular, such activation domains are useful when employed with adeactivated or modified form of the Cas polypeptide with minimizedcleavage activity. In this way, specific recruitment of the Caspolypeptide to the target sequence is enabled by the interacting portionof the target component, and transcriptional activity is controlled bythe activation domains.

Further, transcriptional repressor domains can be employed (e.g., aKrüppel-associated box domain, a SID domain, an Engrailed repressiondomain (EnR), or a SID4X domain). In particular, such repressor domainscan be employed with a deactivated or modified form of the Caspolypeptide with minimized cleavage activity or with an active Caspolypeptide with retained endonuclease activity.

A guiding component may be selected to target any target sequence. Insome embodiments, the target sequence is a sequence within a genome of ahost (e.g., a host cell) or a pathogen (e.g., a pathogen cell). In someembodiments, the guiding component is about or more than about 5, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 35, 40, 45, 50, 75, or more nucleotides in length. In someembodiments, a guiding component is less than about 75, 50, 45, 40, 35,30, 25, 20, 15, 12, or fewer nucleotides in length. The ability of aguiding component to direct sequence-specific binding of a CRISPRcomplex to a target sequence may be assessed by any suitable assay. Forexample, the components of a CRISPR system sufficient to form a CRISPRcomplex, including the guiding component to be tested, may be providedto a host cell having the corresponding target sequence, such as bytransfection with vectors encoding the components of the CRISPRsequence, followed by an assessment of preferential cleavage within thetarget sequence, such as by Surveyor assay. Similarly, cleavage of atarget sequence may be evaluated in a test tube by providing the targetsequence, components of a CRISPR complex, including the guidingcomponent to be tested and a control guiding component different fromthe test guiding component, and comparing binding or rate of cleavage atthe target sequence between the test and control guiding componentreactions.

Outer Layer

The constructs disclosed herein include an outer layer (also referred toas a coating herein) disposed around the core. In particularembodiments, the outer layer includes a combination of lipids supportedby or bonded to the surface of the core. The outer layer or coating mayalso include one or more moieties (e.g., one or more targeting ligands,such as a pegylated lipid). In other embodiments, the outer layer caninclude a polymer layer (e.g., supported by the surface of the core)that can optionally include one or more moieties (e.g., one or moretargeting ligands).

The outer layer or coating, (lipid bilayer) can be characterized by itsthickness (e.g., about 5 nm to about 3 to about 40 nm, such as about 4to about 25 nm, or about 5 to about 15 nm), the number of layers withinthe outer layers (e.g., two, three, four, five, six, seven, or morelipid and/or polymer layers within the outer layer), and/or the netcharge of the outer layer (e.g., a net non-negative charge, such as anet positive charge; or as determined by the composition of the lipidlayer, such as one formed by use of a liposome formulation having morethan about 20 mol. % of a cationic lipid, such as any herein (e.g.,DOTAP)).

In an embodiment, the outer layer includes a cationic lipid, a pegylatedlipid, a zwitterionic lipid, and a sterol, as well as salts of any ofthese (e.g., pharmaceutically acceptable salts).

The lipid layer can include one or more lipids selected from the groupof 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC),1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC),1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC),1,2-dioleoyl-sn-glycero-3-[phosphor-L-serine](DOPS),1,2-dioleoyl-3-trimethylammonium-propane (18:1 DOTAP),1,2-dioleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DOPG),1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE),1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE),1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE),1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC),1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC),1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC),1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC),1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000] (18:1 PEG-2000 PE),1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000] (16:0 PEG-2000 PE),1-oleoyl-2-[12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]lauroyl]-sn-glycero-3-phosphocholine(18:1-12:0 NBD PC),1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy-(polyethyleneglycol)-2000] (DSPE-PEG₂₀₀₀),1-palmitoyl-2-{12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]lauroyl}-sn-glycero-3-phosphocholine(16:0-12:0 NBD PC), a sterol (e.g., cholesterol, desmosterol,diplopterol, cholestanol, cholic acid, 12-deoxycholic acid,7-deoxycholic acid, or a derivative thereof, such as cholesterolsulfate), and mixtures thereof and conjugated forms thereof (e.g.,conjugated to PEG moieties, peptides, polypeptides, includingimmunogenic peptides, proteins and antibodies, and nucleic acids (e.g.,RNA and DNA) by way of a covalent bond or by way of a linker or spacer(e.g., any described herein).

In an embodiment, the outer layer also includes a polymer, including,for example, polyethylene glycol (PEG) or polyethylene oxide (PEO)(e.g., a PEG-polyester), or a copolymer (e.g., a diblock copolymer, suchas an amphiphilic diblock copolymer). Non-limiting polymers include aPEG-lactic acid polymer (PEG-LA, e.g.,poly(ethyleneglycol)-b-poly(lactic acid) copolymer orPEG-b-poly(D,L-lactic acid)); a polycarbonate-polyglutamic acid polymer(PC-PGA, e.g., poly(trimethylene carbonate)-b-poly(glutamic acid); apoly(lactic acid) (PLA, e.g., methoxy poly(ethyleneglycol)-Gly-Phe-Leu-Gly-Phe-poly(D,L-lactide), PEG-PLA, ormaleimide-PEG-PLA); a poly(butadiene) (PBD, e.g., PEO-b-PBD or PEG-PBD);a poly(caprolactone) (PCL, e.g., PEG-PCL, PEO-PCL,PEG-b-poly(ε-caprolactone), mPEG-poly(ε-caprolactone), α-carboxylPEG-poly(3-caprolactone)/PEG-PLA, orPEO-b-poly(γ-methyl-3-caprolactone)); and a PEG- or PEO-polypeptide(e.g., PEG-b-poly(2-hydroxyethyl aspartamide) substituted with octadecylchains, poly(carboxyl ethyleneglycol-γ-glutamate)-co-poly(distearin-γ-glutamate), or poly(ethyleneglycol)-γ-glutamate)-co-poly (distearin-γ-glutamate)).

The outer layer can be a hybrid layer (e.g., including one or morelipids and one or more polymers). Exemplary hybrid layers can include alipid (e.g., any described herein), an optional sterol, and a polymer(e.g., any described herein, such as a polymer including PEG or PEO).

Cores, lipids, polymers, and cargos can be PEGylated with a variety ofpolyethylene glycol-containing compositions as described herein. PEGmolecules can have a variety of lengths and molecular weights andinclude, but are not limited to, PEG 200, PEG 1000, PEG 1500, PEG 2000,PEG 4600, PEG 5000, PEG 10,000, PEG-peptide conjugates or combinationsthereof.

In one instance, the outer layer includes a cationic lipid (e.g.,DOTAP), a zwitterionic lipid (e.g., DOPE), a sterol (e.g., cholesterol),and a PEGylated lipid (e.g., 1,2-distearoyl-sn-157glycero-3-phosphoethanolamine-N-[carboxy-(polyethylene glycol)-2000(DSPE-PEG2000)). These four components may be in a molar ratio of about1 cationic lipid (e.g., DOTAP) to 1 zwitterionic lipid (e.g., DOPE) to0.9 sterol (e.g., cholesterol) to 0.15 PEGylated lipid (e.g.,DSPE-PEG2000). In an instance, each of these ratios may optionally varyby plus or minus 10%, or plus or minus 5%, or plus or minus 3%.Accordingly, each number recited in the ratio above, may range from amultiple of about 0.9 to 1.1, 0.95 to 1.05, or 0.97 to 1.03.

In an instance, the outer layer includes about 10 to about 50 mol. %cationic lipid (e.g., DOTAP), about 10 to 50 mol. % zwitterionic lipid(e.g., DOPE), about 5 to about 45 mol. % sterol (e.g., cholesterol), andabout 2 to 8 mol. % of a PEGylated lipid (e.g., DSPE-PEG2000). Inanother instance, the outer layer includes about 20 to about 40 mol. %cationic lipid (e.g., DOTAP), about 20 to 40 mol. % zwitterionic lipid(e.g., DOPE), about 10 to about 35 mol. % sterol (e.g., cholesterol),and about 2.5 to 6 mol. % of a PEGylated lipid (e.g., DSPE-PEG2000). Inanother instance, the outer layer includes about 30 to about 35 mol. %cationic lipid (e.g., DOTAP), about 30 to 35 mol. % zwitterionic lipid(e.g., DOPE), about 27 to about 33 mol. % sterol (e.g., cholesterol),and about 3 to 5 mol. % of a PEGylated lipid (e.g., DSPE-PEG2000).

In particular embodiments, the outer layer includes about 33 mol % ofthe cationic lipid, about 33 mol % of the zwitterionic lipid, about 30mol % of the sterol, and about 4% of the PEGylated lipid.

Exemplary cationic lipids include1,2-dioleoyl-3-trimethylammonium-propane (DOTAP),1,2-stearoyl-3-trimethylammonium-propane (18:0 TAP),1,2-dipalmitoyl-3-trimethylammonium-propane (16:0 TAP),1,2-dimyristoyl-3-trimethylammonium-propane (14:0 TAP),1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA),N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-amino-propyl)amino]butylcarboxamido)ethyl]-3,4-di[oleyloxy]-benzamide(MVL5), ethylphosphocholine (ethyl PC) (e.g.,1,2-dimyristoleoyl-sn-glycero-3-ethylphosphocholine,1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine,1,2-dioleoyl-sn-glycero-3-ethylphosphocholine,1,2-distearoyl-sn-glycero-3-ethylphosphocholine,1,2-dipalmitoyl-sn-glycero-3-ethylphosphocholine,1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine, or1,2-dilauroyl-sn-glycero-3-ethylphosphocholine),dimethyldioctadecylammonium (DDAB),1,2-dipalmitoyl-sn-glycero-O-ethyl-3-phosphocholine (EDPPC), or anydescribed herein.

Exemplary zwitterionic lipids include DOPC, DPPC, DOPE, DPPE, POPC,DLPC, DSPC, DMPC, SOPC, or any described herein.

Exemplary, non-limiting sterols include cholesterol (e.g., from ovinewool or from plant sources), campestanol, campesterol, cholestanol,cholestenone, desmosterol, 7-dehydrodesmosterol, dehydroepiandrosterone(DHEA), desmosterol, diosgenin, FF-MAS(14-demethyl-14-dehydrolanosterol), lanosterol, lathosterol,pregnenolone, sitostanol, sitosterol, stigmasterol, zymosterol,zymostenol, zymosterone, as well as derivatives thereof, such assulfates thereof, esters thereof, stereoisomers thereof, deuteratedforms thereof, sulfonated forms thereof, phosphorylated forms thereof,unsaturated forms thereof, keto forms thereof, oxidized forms thereof,an oxysterol thereof, PEGylated forms thereof (e.g.,cholesterol-(polyethylene glycol-600)), or substituted forms thereof(e.g., having one or more hydroxyl, epoxy, alkyl, phospho, and/or halo,such as fluoro).

Exemplary PEGylated lipids (e.g., a lipid having a poly(ethylene glycolmoiety)) include PEGylated DSPE (e.g.,1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethyleneglycol)-X] (DSPE X) or N-[carbonyl-2′,3′-bis(methoxypolyethyleneglycolX)]-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE-2arm PEGX)),PEGylated phosphoethanolamine (PE) (e.g.,1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-X] (18:1 PEGX PE),1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-X] (18:0 PEGX PE),1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-X](14:0 PEGX PE), or1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-5000] (16:0 PEGX PE)), PEGylated DPPE (e.g.,N-(carbonyl-methoxypolyethyleneglycolX)-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine), PEGylated DMPE(e.g., N-(carbonyl-methoxypolyethyleneglycolX)-1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine), PEGylated DPG(e.g., 1,2-dipalmitoyl-sn-glycerol, methoxypolyethylene glycol),PEGylated DSG (e.g., 1,2-distearoyl-sn-glycerol, methoxypolyethyleneglycol), PEGylated DOG (e.g., 1,2-dioleoyl-sn-glycerol,methoxypolyethylene glycol), or PEGylated DMG (e.g.,1,2-dimyristoyl-sn-glycerol, methoxypolyethylene glycol), where Xindicates an approximate number average molecular weight (Mn) asmeasured by Gel Permeation Chromotography (GPC) with appropriatestandards, and where X is 500, 3000, 2000, 1000, 750, 550, or 350.

The outer layer of the particle can be composed of lipids, polymers,and/or components in an amount similar to that provided by the lipidformulation. For instance, an exemplary lipid formulation comprisingabout 47 mol. % of a cationic lipid can provide a lipid layer (for aconstruct) that comprises 47 mol. % of that cationic lipid. Thus, anycomposition provided for a lipid formulation herein also provides acomposition for the outer layer.

Targeting Ligands

Optionally the construct can include one or more cell targeting species,cell receptor ligands, cell penetrating peptides, fusogenic peptides,and/or targeting peptides. Such species can be included within thecargo, configured to be expressed by a plasmid of the cargo, locatedwithin the outer layer, and/or provided by an external surface of theouter layer (e.g., provided by the outer lipid layer). In an embodimentthe targeting ligand can be added to the construct via the pegylatedlipid, PEG2000 and derivatives thereof. The composition of the outerlayer can include one or more components that facilitate ligandorientation, maximize cellular interaction, provide lipid stability,and/or confer enhanced cellular entry.

In some instances, the targeting ligand can be a cell penetrationpeptide, a fusogenic peptide, or an endosomolytic peptide, which arepeptides that aid a particle in translocating across a lipid bilayer,such as a cellular membrane or endosome lipid bilayer of the host cell.In one embodiment, the targeting ligand is optionally crosslinked onto alipid layer surface of the outer layer.

Endosomolytic peptides are a sub-species of fusogenic peptides asdescribed herein. Representative and preferred electrostatic cellpenetration (fusogenic) peptides include an 8 mer polyarginine(NH₂—RRRRRRRR—COOH, SEQ ID NO:1), among others known in the art, whichare included in or on particles in order to enhance the penetration ofinto cells. Representative endosomolytic fusogenic peptides(“endosomolytic peptides”) include H5WYG peptide(NH₂-GLFHAIAHFIHGGWHGLIHGWYGGC-COOH, SEQ ID NO:2), RALA peptide(NH₂-WEARLARALARALARHLARALARALRAGEA-COOH, SEQ ID NO:3), KALA peptide(NH₂-WEAKLAKALAKALAKHLAKALAKALKAGEA-COOH), SEQ ID NO:4), GALA(NH₂-WEAALAEALAEALAEHLAEALAEALEALAA-COOH, SEQ ID NO:5) and INF7(NH₂-GLFEAIEGFIENGWEGMIDGWYG-COOH, SEQ ID NO:6), or fragments thereof,among others. In one instance, the targeting ligand includes an aminoacid sequence having at least 80% sequence identity (e.g., at least 85%,90%, 95%, or 99% sequence identity) to any one of SEQ ID NOs:1-6, or afragment thereof.

Proteins gain entry into the nucleus through the nuclear envelope. Yetother ligands can include a nuclear localization sequence (NLS), e.g.,N112-GNQSSNFGPMKGGNFGGRSSGPY GGGGQYFAKPRNQGGYGGC-COOH (SEQ ID NO:9),RRMKWKK (SEQ ID NO:10), PKKKRKV (SEQ ID NO:11), and KR[PAATKKAGQA]KKKK(SEQ ID NO:12), the NLS of nucleoplasmin, a prototypical bipartitesignal comprising two clusters of basic amino acids, separated by aspacer of about 10 amino acids. Numerous other nuclear localizationsequences are well known in the art. See, for example, LaCasse E C etal., “Nuclear localization signals overlap DNA- or RNA-binding domainsin nucleic acid-binding proteins,” Nucl. Acids Res. 1995; 23:1647-56;Weis, K, “Importins and exportins: how to get in and out of thenucleus,” [published erratum appears in Trends Biochem. Sci. 1998 July;23(7):235] Trends Biochem. Sci. 1998; 23:185-9; and Cokol M et al., EMBORep. 2000 Nov. 15; 1(5): 411-5, each of which is incorporated herein byreference in its entirety.

Preferred ligands which may be used to target cells include peptides,affibodies, and antibodies (including monoclonal and/or polyclonalantibodies). In certain embodiments, targeting ligands selected from thegroup consisting of Fcγ from human IgG (which binds to Fcγ receptors onmacrophages and dendritic cells), human complement C3 (which binds toCR1 on macrophages and dendritic cells), ephrin B2 (which binds to EphB4receptors on alveolar type II epithelial cells), SP94 peptide (whichbinds to unknown receptor(s) on hepatocyte-derived cells), and METreceptor binding peptide. Exemplary, non-limiting SP94 peptides includeSP94 free peptide (H2N-SFSIILTPILPL-COOH, SEQ ID NO:126), a SP94 peptidemodified with C-terminal Cys for conjugation (H2N-SFSIILTPILPLGGC-COOH,SEQ ID NO:127), and a further modified SP94 peptide(H2N-SFSIILTPILPLEEEGGC-COOH, SEQ ID NO:128). Exemplary MET bindingpeptides include ASVHFPP (SEQ ID NO:121), TATFWFQ (SEQ ID NO:122),TSPVALL (SEQ ID NO:123), IPLKVHP (SEQ ID NO:124), and WPRLTNM (SEQ IDNO:125).

Other exemplary targeting ligands include poly-L-arginine, including(R)_(n), where 6<n<12, such as an R12 peptide (e.g., RRRRRRRRRRRR (SEQID NO:210)) or an R9 peptide (e.g., RRRRRRRRR (SEQ ID NO:211)); apoly-histidine-lysine, such as a (KH)₉ (e.g., KHKHKHKHKHKHKHKHKH (SEQ IDNO:212)); a Tat protein or derivatives and fragments thereof, such asRKKRRQRRR (SEQ ID NO:213), GRKKRRQRRRPQ (SEQ ID NO:214), GRKKRRQRRR (SEQID NO:215), GRKKRRQRRRPPQ (SEQ ID NO:216), YGRKKRRQRRR (SEQ ID NO:217),and RKKRRQRRRRKKRRQRRR (SEQ ID NO:218); a Cady protein or derivativesand fragments thereof, such as Ac-GLWRALWRLLRSLWRLLWRA-cysteamide (SEQID NO:219); a penetratin protein or derivatives and fragments thereof,such as RQIKIWFQNRRMKWKKGG (SEQ ID NO:220), RQIRIWFQNRRMRWRR (SEQ IDNO:221), and RQIKIWFQNRRMKWKK (SEQ ID NO:222); an antitrypsin protein orderivatives and fragments thereof, such as CSIPPEVKFNKPFVYLI (SEQ IDNO:223); a temporin protein or derivatives and fragments thereof, suchas FVQWFSKFLGRIL-NH₂ (SEQ ID NO:224); a MAP protein or derivatives andfragments thereof, such as KLALKLALKALKAALKLA (SEQ ID NO:225); a RWprotein or derivatives and fragments thereof, such as RRWWRRWRR (SEQ IDNO:226); a pVEC protein or derivatives and fragments thereof, such asLLIILRRRIRKQAHAHSK (SEQ ID NO:227); a transportan protein or derivativesand fragments thereof, such as GWTLNSAGYLLGKIN LKALAALAKKIL (SEQIDNO:228); a MPG protein or derivatives and fragments thereof, such asGALFLGFLGAAGSTMGAWSQPKKKRKV (SEQ ID NO:229); a Pep protein orderivatives and fragments thereof, such as KETWWETWWTEWSQPKKKRKV (SEQ IDNO:230), Ac-KETWWETWWTEWSQPKKKRKV-cysteamine (SEQ ID NO:231), andWKLFKKILKVL-amide (SEQ ID NO:232); a Bp100 protein or derivatives andfragments thereof, such as KKLFKKILKYL (SEQ ID NO:233) andKKLFKKILKYL-amide (SEQ ID NO:234); a maurocalcine protein or derivativesand fragments thereof, such as GDC(acm)LPHLKLC (SEQ ID NO:235); acalcitonin protein or derivatives and fragments thereof, such asLGTYTQDFNKFHTFPQTAIGVGAP (SEQ ID NO:236); a neurturin protein orderivatives and fragments thereof, such asGAAEAAARVYDLGLRRLRQRRRLRRERVRA (SEQ ID NO:237); and a human P1 proteinor derivatives and fragments thereof, such asMGLGLHLLVLAAALQGAWSQPKKKRKV (SEQ ID NO:238).

In one instance, the targeting ligand includes an amino acid sequencehaving at least 80% sequence identity (e.g., at least 85%, 90%, 95%, or99% sequence identity) to any one of SEQ ID NOs:10-12 and 210-238 or afragment thereof (e.g., having a length of about 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, 26, 28, 30, 32, 34, 36,38, 40, or more amino acids).

Exemplary ligands also include a peptide that binds to ephrin B2, totarget Vero cells; Fcγ to target THP-1 cells and primary alveolarmacrophages; the ‘GE11’ peptide (see, e.g., Li Z et al., FASEB J 2006;19: 1978-85) to target A549 cells and primary alveolar epithelial cells;the ‘SP94’ peptide (see, e.g., Lo A et al., Molec. Cancer Therap. 2008;7:579-89) to target HepG2 cells and primary hepatocytes; humancomplement C3, which binds to receptors on macrophages and dendriticcells; or the ‘H5WYG’ peptide, which ruptures the membranes of acidicintracellular vesicles via the ‘proton sponge’ mechanism (see, e.g.,Moore N M et al., J. Gene. Med. 2008 10: 1134-49).

Other ligands include a peptide (e.g., a peptide zip code or a cellpenetrating peptide), an endosomolytic peptide, an antibody (includingfragments thereof), affibodies, a carbohydrate, an aptamer, a cluster ofdifferentiation (CD) protein, or a self-associated molecular pattern(SAMP) (e.g., as described in Lambris J D et al., Nat. Rev. Microbiol.2008; 6(2):132; and Poon I K H, Cell Death Differ. 2010; 17:381-97, eachof which is incorporated herein by reference in its entirety). ExemplaryCD proteins include CD47 (OMIM Entry No. 601028, a marker of self thatallows RBC to avoid phagocytosis), CD59 (OMIM Entry No. 107271, a markerthat prevents lysis by complement), C1 inhibitor (C1INH, OMIM Entry No.606860, a marker that suppresses activation of the host's complementsystem), CD200 (OMIM Entry No. 155970, an immunosuppressive factor),CD55 (OMIM Entry No. 125240, a marker that inhibits the complementcascade), CD46 (OMIM Entry No. 120920, a marker that inhibits thecomplement cascade), and CD31 (OMIM Entry No. 173445, an adhesionregulator and a negative regulator of platelet-collagen interactions).Each recited OMIM Entry is incorporated herein by reference in itsentirety.

Other useful ligands can be employed, such as those identified by the‘BRASIL’ (Biopanning and Rapid Analysis of Selective InteractiveLigands) method (see, e.g., Giordano R J et al., Nat. Med. 2001;7:1249-53; Giordano R J et al., Proc. Natl Acad. Sci. USA 2010;107(11):5112-7; and Kolonin M G et al., Cancer Res. 2006; 66:34-40) toidentify novel targeting peptides and single-chain variable fragments(scFvs) via phage display (see, e.g., Giordano R J et al., Chem. Biol.2005; 12:1075-83; Giordano R J et al., Proc. Natl Acad. Sci. USA 2010;107(11):5112-7; Kolonin M G et al., Cancer Res. 2006; 66:34-40; TonelliR R et al., PLoS Negl. Dis. 2010; 4:e864; Lionakis M S et al., Infect.Immun. 2005; 73:7747-58; and Barbu E M et al., PLoS Pathog. 2010;6:e1000726).

Particle Characteristics and Surface Properties

The construct (or particle) can be characterized by, e.g., overallcharge, dimension, or dispersity. In some embodiments, one or moreoptional targeting ligands can be present in or on an outer layer. Theparticle can have a diameter, circumference, length, width, height, etc.Exemplary values for dimensions include, without limitation, greaterthan about 10 nm (e.g., greater than about 20 nm, 30 nm, 40 nm, 50 nm,60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 125 nm, 150 nm, 200 nm, 300 nm, 500nm, 750 nm, 1 m, 2 m, 5 m, 10 m, 20 m) or of about 2 nm to 500 nm (e.g.,from 2 nm to 50 nm, 2 nm to 100 nm, 2 nm to 150 nm, 2 nm to 200 nm, 2 nmto 300 nm, 2 nm to 400 nm, 10 nm to 50 nm, 10 nm to 100 nm, 10 nm to 150nm, 10 nm to 200 nm, 10 nm to 300 nm, 10 nm to 400 nm, 10 nm to 500 nm,20 nm to 50 nm, 20 nm to 100 nm, 20 nm to 150 nm, 20 nm to 200 nm, 20 nmto 300 nm, 20 nm to 400 nm, 20 nm to 500 nm, 50 nm to 100 nm, 50 nm to150 nm, 50 nm to 200 nm, 50 nm to 300 nm, 50 nm to 400 nm, 50 nm to 500nm, 100 nm to 150 nm, 100 nm to 200 nm, 100 nm to 300 nm, 100 nm to 400nm, 100 nm to 500 nm, 150 nm to 200 nm, 150 nm to 300 nm, 150 nm to 400nm, 150 nm to 500 nm, 200 nm to 300 nm, 200 nm to 400 nm, or 200 nm to500 nm). In each case, the dimension of particle or construct is largerthan the core dimension of the same type.

In particular embodiments, a plurality of particles is monodisperse indiameter, such as by having a polydispersity index (PdI) that is lessthan about 0.2 or by having a PdI that is of about 0.05 to about 0.2(e.g., from 0.05 to 0.1, 0.05 to 0.15, 0.1 to 0.15, 0.1 to 0.2, or 0.15to 0.2). The calculations used for the determination of size and PDIparameters are defined in the ISO standard documents 13321:1996 E andISO 22412:2008. In some embodiments, the monodisperse particles range ina size of from about 50 nm to about 475 nm (e.g., from 150 nm (+/−10 nm)to 350 nm (+/−15 nm)).

In embodiments, the particle (or a plurality of particles) has a charge(or a net charge) that is near neutral (e.g., a zeta potential of about+5 mV to −5 mV, or about +10 to about −10 mV). As mentioned above, theconstruct can include appropriate targeting ligands to promote theircell-specific binding and internalization, and can include a usefulligand to promote endosomal escape or nuclear localization within hostcells.

Compositions and Formulations

The present constructs can be formulated, for example, for subcutaneous(SC), intranasal (IN), aerosol, intravenous (IV), intramuscular (IM),intraperitoneal (IP), oral, topical, transdermal, or retro-orbitaldelivery. Exemplary dosages include, e.g., about 0.01 g (construct)/kg(body wt.) to about 0.2 g/kg, such as, 0.05 g/kg to about 0.15 g/kf, orabout 0.07 to 0.1 g/kg. A dose of 0.1 g/kg was well tolerated in miceand could translate to humans. A dose of 0.1 g LC-MSNs/kg/day was testedup to four days in mice. Daily doses can be given from 2 to 10 days,such as 2 to 8, or 3 to 5 days. Certain cargos may vary the dosage, forexample, RNP delivery may be applied only up to four days.

The formulation or composition can include a plurality of particles(e.g., an effective amount thereof) and an optional pharmaceuticallyacceptable excipient (e.g., any described herein). In some instances,the pharmaceutical composition includes a population of particles (e.g.,any described herein) in an amount effective for modulating or modifyinga target gene within a subject in combination with a pharmaceuticallyacceptable carrier, additive, or excipient. In other instances, thecomposition further includes a drug, a therapeutic agent, etc., which isnot disposed as cargo within the particle.

The composition can be formulated in any useful manner with a pluralityof particles. Such formulations can be included with a medium, excipient(e.g., lactose, saccharide, carbohydrate, mannitol, leucine, PEG, ortrehalose), additive, propellant, solution (e.g., aqueous solution, suchas a buffer), additive, preservative, carrier (e.g., aqueous saline,aqueous dextrose, glycerol, or ethanol), binder (e.g., saccharide,cellulose preparation, starch paste, or methyl cellulose), filler, ordisintegrator.

Pharmaceutical compositions according to the present disclosure includean effective population of constructs herein formulated to effect anintended result (e.g., immunogenic result, therapeutic result and/ordiagnostic analysis, including the monitoring of therapy) formulated incombination with a pharmaceutically acceptable carrier, additive, orexcipient. The particles within the population of the composition may bethe same or different depending upon the desired result to be obtained.Pharmaceutical compositions according to the present disclosure may alsocomprise an addition bioactive agent or drug, such as an antibiotic orantiviral agent.

Formulations and compositions containing the particles according to thepresent disclosure may take the form of liquid, solid, semi-solid orlyophilized powder forms, such as, for example, solutions, suspensions,emulsions, sustained-release formulations, tablets, capsules, powders,suppositories, creams, ointments, lotions, aerosols, or patches, in unitdosage forms suitable for simple administration of precise dosages.

Methods for preparing such dosage forms are known or apparent to thoseskilled in the art; for example, see Remington's Pharmaceutical Sciences(17th Ed., Mack Pub. Co., 1985). The composition to be administered willcontain a quantity of the selected compound in a pharmaceuticallyeffective amount for therapeutic use in a biological system, including apatient or subject according to the disclosed technology.

Methods

The constructs herein can be adapted to recognize the target and, ifneeded, deliver the one or more cargos to treat that target. Exemplarytargets include a cell, a pathogen, an organ (e.g., dermis, vasculature,lymphoid tissue, liver, lung, spleen, kidneys, heart, brain, bone,muscle, etc.), a cellular target (e.g., targets of the subject, such asa human subject, including host tissue, host cytoplasm, host nucleus,etc., in any useful cell, such as e.g., hepatocytes, alveolar epithelialcells, and innate immune cells, etc.); as well as targets for exogenouscells and organisms, such as extracellular and/or intracellularcomponents of a pathogen, e.g., bacteria), a molecular target (e.g.,within the subject or the exogenous cell/organism, such as pathogen DNA,host DNA, pathogen RNA, pathogen proteins, surface proteins orcarbohydrates of any subject or exogenous cell).

In one instance, the particle is employed to target a host (e.g., asubject), a pathogen, or both (e.g., thereby treating the subject and/orthe target). Exemplary pathogens include a bacterium, such as Bacillus(e.g., B. anthracis), Enterobacteriaceae (e.g., Salmonella, Escherichiacoli, Yersinia pestis, Klebsiella, and Shigella), Yersinia (e.g., Y.pestis or Y. enterocolitica), Staphylococcus (e.g., S. aureus),Streptococcus, Gonorrheae, Enterococcus (e.g., E. faecalis), Listeria(e.g., L. monocytogenes), Brucella (e.g., B. abortus, B. melitensis, orB. suis), Vibrio (e.g., V. cholerae), Corynebacterium diphtheria,Pseudomonas (e.g., P. pseudomallei or P. aeruginosa), Burkholderia(e.g., B. mallei or B. pseudomallei), Shigella (e.g., S. dysenteriae),Rickettsia (e.g., R. rickettsii, R. prowazekii, or R. typhi),Francisella tularensis, Chlamydia psittaci, Coxiella burnetii,Mycoplasma (e.g., M. mycoides), etc.; mycotoxins, mold spores, orbacterial spores such as Clostridium botulinum and C. perfringens; avirus, including DNA or RNA viruses, such as Adenoviridae (e.g.,adenovirus), Arenaviridae (e.g., Machupo virus), Bunyaviridae (e.g.,Hantavirus or Rift Valley fever virus), Coronaviridae (e.g.,SARS-Cov-2), Orthomyxoviridae (e.g., influenza viruses), Filoviridae(e.g., Ebola virus and Marburg virus), Flaviviridae (e.g., Japaneseencephalitis virus, hepatitis C virus, and Yellow fever virus),Hepadnaviridae (e.g., hepatitis B virus), Herpesviridae (e.g., herpessimplex viruses, herpesvirus, cytomegalovirus, Epstein-Barr virus, orvaricella zoster viruses), Papillomaviridae (e.g., papilloma viruses),Papovaviridae (e.g., papilloma viruses), Paramyxoviridae (e.g.,respiratory syncytial virus, measles virus, mumps virus, orparainfluenza virus), Parvoviridae, Picornaviridae (e.g., poliovirusesand hepatitis A virus), Polyomaviridae, Poxviridae (e.g., variolaviruses or vaccinia virus), Reoviridae (e.g., rotaviruses), Retroviridae(e.g., human T cell lymphotropic viruses (HTLV) and humanimmunodeficiency viruses (HIV)), Rhabdoviridae (e.g., rabies virus), andTogaviridae (e.g., encephalitis viruses, yellow fever virus, and rubellavirus)); a protozoon, such as Cryptosporidium parvum, Encephalitozoa,Plasmodium, Toxoplasma gondii, Acanthamoeba, Entamoeba histolytica,Giardia lamblia, Trichomonas vaginalis, Leishmania, or Trypanosoma(e.g., T. brucei and T. Cruzi); a helminth, such as cestodes(tapeworms), trematodes (flukes), or nematodes (roundworms, e.g.,Ascaris lumbricoides, Trichuris trichiura, Necator americanus, orAncylostoma duodenale); a parasite (e.g., any protozoa or helminthsdescribed herein); or a fungus, such as Aspergilli, Candidae,Coccidioides immitis, and Cryptococci. Other pathogens include amulti-drug resistant (MDR) pathogen, such as MDR forms of any pathogendescribed herein. Additional pathogens are described in Cello J et al.,Science 2002; 297:1016-8; Gibson D G et al., Science 2010; 329:52-6;Jackson R J et al., J. Virol. 2001; 75:1205-10; Russell C A et al.,Science 2012; 336:1541-7; Tumpey T M et al., Science 2005; 310:77-80;and Weber N D et al., Virology 2014; 454-455c:353-61, each of which isincorporated herein by reference in its entirety.

The constructs of the disclosed technology can be employed to treat anyuseful disease that would benefit from genetic knock-out of a knownprotein. For instance, the particles can be employed to treat a subjectfrom a disease correlated with the presence of that known protein (e.g.,a known protein expressed within the subject or within a pathogeninfecting that subject). Other diseases include a genetic disorder(e.g., Huntington's disease, hemophilia, sickle cell anemia, metabolicdisorders, etc.), in which expression of a known protein is correlatedwith the disease or its symptoms.

The constructs can be employed to transform a subject (e.g., bygenetically modifying a target gene within the subject by employing aCRISPR component configured to bind to that target gene). Thus, in oneinstance, the particle can be configured to bind to a target sequence ina genomic sequence of the subject in order to modulate that targetsequence. Modulation can include activating, inactivating, deactivating,and/or modifying expression or activity of the target sequence. Forexample, the cargo can bind to the target sequence, e.g., therebyinhibiting expression of one or more proteins encoded by the targetsequence. In another example, the cargo cleaves the target sequence andoptionally inserts a further nucleic acid sequence into the genomicsequence of the subject. In yet another example, the cargo activates thetarget sequence. Any useful target sequence can be modulated.

Methods of treating patients or subjects in need for a particulardisease state or infection can include administration an effectiveamount of a pharmaceutical composition having a plurality of constructs(e.g., any described herein). Additional methods include diagnosticmethods, which can include administering an effective amount of apopulation of diagnostic particles to a subject in need thereof. In someembodiments, the population of particles, or a portion thereof, includesa ligand (e.g., to bind to target cells) and a reporter (e.g., toindicate binding to the target cell), whereupon the binding of one ormore particles to cells as evidenced by the reporter component (moiety)will enable a diagnosis of the existence of a disease state in thesubject.

In accordance with the present disclosure, there may be employedconventional molecular biology, microbiology, and recombinant DNAtechniques within the skill of the art. Such techniques are explainedfully in the literature. See, e.g., Sambrook et al, 2001, “MolecularCloning: A Laboratory Manual”; Ausubel, ed., 1994, “Current Protocols inMolecular Biology” Volumes I-III; Celis, ed., 1994, “Cell Biology: ALaboratory Handbook” Volumes I-III; Coligan, ed., 1994, “CurrentProtocols in Immunology” Volumes I-III; Gait ed., 1984, “OligonucleotideSynthesis”; Hames & Higgins eds., 1985, “Nucleic Acid Hybridization”;Hames & Higgins, eds., 1984, “Transcription And Translation”; Freshney,ed., 1986, “Animal Cell Culture”; IRL.

The present disclosure also relates to methods of fabricating aconstruct (e.g., or a population of particles). The method can include,e.g., providing a core (including a plurality of cores) having anyuseful characteristic (e.g., any described herein, such as having adimension greater than about 50 nm, having a negative charge, having oneor more pores, and/or including a silica); incubating the core with oneor more cargo (e.g., any herein, including a plasmid, a CRISPRcomponent, etc.), thereby providing a loaded core; and exposing theloaded core to a lipid formulation (e.g., any described herein).

In other embodiments, the method can include providing a core and thenexpanding the pores present on the core. In some instance, a method caninclude: providing a core including an external surface and a pluralityof pores in fluidic communication with the external surface (e.g., wherean average dimension of the plurality of pores is characterized by afirst dimension); expanding the pores (e.g., thereby providing a corecomprising a plurality of expanded pores, wherein an average dimensionof the plurality of expanded pores is characterized by a seconddimension that is greater than the first dimension); incubating the corewith one or more cargo, thereby providing a loaded core; and exposingthe loaded core to a polymer formulation or a lipid formulation to forman outer layer supported upon the external surface of the core (e.g.,thereby providing the construct).

FIG. 3A-3C shows exemplary methods for providing a construct and its usefor in vitro gene editing. Provided are schematics of (A) an exemplarymethod for loading RNP within a construct and (B) an exemplary methodfor in vitro gene editing by use of an RNP-loaded construct. Alsoprovided are (C) fluorescence photomicrographs showing delivery of anRNP-loaded construct to a reporter cell line with an AAVS1 target site,in which effective gene-editing by the RNP results in a frameshiftmutation and GFP expression.

EXAMPLES

Numerous experiments were run with various LCMSN (lipid-coatedmesoporous nanoparticle) constructs comprising lipid compositions andmesoporous nanoparticles (MSNs) until a surprisingly effectivecombination was found. In particular, a stellate MSN with a 33 mol %,DOTAP, 33 mol % DOPE, 30 mol % cholesterol and 4 mol % DSPE-PEG2000synthesized in Example 3 below was chosen after trial and errorexperimentation to achieve surprisingly effective delivery ofCRISPR-Cas9 RNP packaged in LC-MSN. To assess the LC-MSN with theselected coating, negative staining and cryo-EM was performed onCRISPR-Cas9 RNP-loaded LC-MSN. A complete lipid coat was visible onmicroscopic analysis. Further details are provided below.

Example 1: MSN Fabrication and Characterization

In Example 1A Stellate MSN synthesis was carried out in a 50 mL roundbottom flask, by combining triethanolamine (70 mg), CTATos (0.300 g, cas#138-32-9) with 20 mL water (Sigma). The solution was stirred at 75-80°C. for 30 min to ensure complete dissolution. Condensation was achievedby adding TEOS (2.9 mL) dropwise to the solution over 5 minutes stirringat 850 rpm. The reaction was then carried out for 2 hours at 75-80° C.with a condenser. MSN solution was removed from heat and cooled for 15minutes then spun down for 15 minutes at 50,000×g. The collected MSNwere washed twice by pure then 190 proof ethanol. Surfactant removal wascarried out by suspending the MSN in 5% v (volume) HCl (12 N, 37%) inethanol in a 100 mL round bottom flask and refluxing for 2 hours (usinga cooling column, refrigerant). This was repeated with HCl 1% instead of5% and then 190 proof ethanol then pure ethanol. The resulting MSN wereresuspended in pure ethanol and passed through a 1 μm filter to removeany aggregates.

Size and zeta potential were assessed using a Zetasizer instrument(Malvern Instruments, Ltd). Morphology was assessed by TEM (JEOL 2010).

In addition, hexagonal prism small pore particles were prepared aspreviously described (LaBauve, et al, Lipid-Coated Mesoporous SilicaNanoparticles for the Delivery of the ML336 Antiviral to InhibitEncephalitic Alphavirus Infection, Sci Rep. 2018; 8: 13990, 2018 Sep.18. doi: 10.1038/s41598-018-32033-w) incorporated herein by reference.These were stored in pure ethanol and quantified. The average size(diameter) by DLS was 160 nm and the zeta potential was −37 mV.

Example 2: Preparation of Liposomes

A 7.27 mg/mL liposome solution in 50:50 PBS:water (4 mM MgCl₂) composedof 33 mol % 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) 33 mol %1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) 30 mol %Cholesterol and 4 mol %1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy-(polyethyleneglycol)-2000](DPSE-PEG2000) (Avanti Polar Lipids) was used. Stock lipids were dilutedin chloroform (Sigma) and 7.27 mg total lipids were combined in a glassscintillation vial then dried down using a rotary evaporator (BuchiCorp.). The resulting lipid films were placed under vacuum overnight toensure complete solvent removal then resuspended in 1 mL 0.5×PBS 4 mMMgCl₂ solution. The suspension was placed in a sonication bath (Branson)and sonicated for 30 minutes at 30° C., then immediately extruded with21 passes through a 100 nm filter (Whatman). Liposome size was assessedby DLS. See FIG. 18B, column labeled “lipid”.

Example 3: Cargo Loading and LC-MSN Fabrication

100 μg of MSN stored in ethanol was spun down in a 1.5 mL Eppendorf tubeat 21,000×g for 10 minutes and washed with 1 mL of water undersonication. Washed MSN were collected by centrifugation and resuspendedin 10 μL of water. CRISPR-Cas-9/gRNA complex from ribonucleoproteins(RNPs) was complexed in 53.3 μL 100 mM NaCl, 50 mM Tris, 10% glycerol,at pH 8.0 by adding 20 pg of SpyCas9 and 6.67 μg sgRNA (a 1:3 ratiogRNA:Cas9) for a final Cas-9 concentration of 0.375 μg/uL and incubatedat 30° C. for 15 minutes to achieve a ratio of 1:5 Cas9:MSN. The RNPsolution was mixed with the MSN with pipetting and sonication thenincubated at room temperature for 30 minutes. Liposomes were fused toRNP loaded MSN by adding 100 μL of extruded liposomes in a mix of 50:50PBS:water supplemented with 4 mM MgCl₂ and pipetting with occasionalsonication. The resulting LC-MSN were collected by centrifugation for 15minutes at 15,000×g and washed with 1 mL PBS then resuspended in 100 μLPBS with pipetting and sonication.

FIG. 18D shows comparison of the RNP-loaded MSN with and without lipids.FIG. 18C shows size measurements during LC-MSN assembly.

Example 4: LC-MSN Loading

Immediately after LC-MSN formation, 25 μg of LC-MSN in PBS was collectedin 1.5 mL Eppendorf tubes. Either a 1.8% solution of TX-100 in PBS, orPBS (12.5 μL) was added and the solution was incubated at 37° C. shakingat 400 rpm for 4 hours. The LC-MSN was then spun down at 21,000×g for 10minutes to separate the supernatant from remaining MSN. The pellets wereresuspended in 37.5 μL of PBS and 4× Laemmli buffer was added and allsamples were boiled for 10 minutes. Samples were run on a 10%polyacrylamide gel (Biorad) with standard known amounts on Cas-9protein.

Loading and release was assessed by densitometry. See FIG. 16 . Westernblot gel images were taken and analyzed by ImageJ software where thedensity of signal across protein bands were quantified and normalized toCas9 protein standards.

Example 5: Reporter Cell Line

The reporter cell line was generating in A549 cells (ATCC) using afluorescent reporter system from PNA Bio. The gene was cloned into alentivirus vector, and particles derived from a single clone were usedto transduce human A549 lung epithelial cells. The transduced cellsconstitutively express red fluorescent protein (RFP). The RFP signal islinked to a gene encoding green fluorescent protein (EGFP) gene.Expression of EGFP is dependent on double strand breaks that lead to aframe-shift mutation. See FIG. 19 illustrating this Example. Reporterand parental cell lines were maintained in F-K12 media (Gibco)supplemented with 10% FBS (Gibco) and penicillin/streptomycin.

Example 6: In Vitro Editing Experiments

Cells were seeded at 50,000 cells per well in 12-well plates (Costar) in1 mL of F-K12 media supplemented with 5% FBS (Gibco) and pen/strep(Gibco) and were incubated overnight. The RNP loaded LC-MSNs wereresuspended in PBS at 1 mg/mL. LC-MSNs were added directly to the 1 mLof media in each well at increasing concentrations of 20 μg/mL, 40μg/mL, 60 μg/mL and 80 μg/mL. Media was replaced after 16 hours andediting efficiency was assessed at 72 hours by microscopy and flowcytometry.

Example 7: Flow Cytometry

Editing was assessed by flow cytometry. Cells in 12-well plates werelifted with 250 μL trypsin (Gibco) collected by centrifugation at 4000×gin 1.5 mL tubes, washed with 1 mL PBS (Gibco) and resuspended in 1 mL ofPBS supplementing with 4% paraformaldehyde. Fixed cells were assessedwith an Accuri C3 flow cytometer (BD). Untreated and RNP-CRISPR Maxtreated A549R cells were used as negative and positive controlsrespectively for gating.

Example 8: Cryo-EM Analysis

For cryo-EM analysis, freshly prepared RNP loaded and unloaded LC-MSNswere vitrified using an automatic plunge freezer EM (Leica). 4 μL ofLC-MSN solution was added to a C-flat grid (Protochips, Inc.) with 2 μmholes and blotted with filter paper. The grid was plunged into liquidethane for flash freezing. Frozen grids were stored in liquid nitrogenand transferred to a JEM 2200FS electron microscope (JEOL Ltd.). Gridswere imaged at 200 keV using DE-20 (Direct Detector Inc.) directelectron detector camera. The energy selecting slit was set to 20 eV andthe microscope had a field emission electron source and omega-typeelectron energy filter to remove inelastically scattered electrons fromthe image formation.

A DE-20 camera was used to collect images in movie mode with a framerate of 25 frames/sec. After image collection, frame alignment wasperformed using the E_process_frames.py script provided by DirectElectron Inc. Images were collected at 40,000× magnification and thepixel size on the specimen scale corresponded to 1.5 A/pixel. See FIG.14C.

Example 9: Phosphotungstic Acid-Based Negative Staining of LC-MSN forTEM

RNP-loaded LC-MSNs were prepared as described above, diluted in PBS to0.1 mg/mL then added (5 μL) to a TEM copper grid (Sigma Aldrich). Aftera 5 minute drying period, a phosphotungstinic acid solution (2% inwater) was added (5 μL) to the grid and removed by dabbing with thecorner of a Kimwipe (Kimtech) after 10-15 seconds. The grid was washedwith 15 μL water and allowed to dry at room temperature prior to TEMimaging as described above. See FIG. 14B.

Example 10: In Vivo Delivery of CRISPR-Cas9 RNP LC-MSN

To assess the efficiency of the LC-MSN delivery vehicle in vivo a murineliver NPC was utilized.

Example 11: Comparison of Different Core Particles

Delivery of the CRISPR-Cas-9 cargo was tested in vitro with varyingtypes of particles (cores) that were able to form an LC-MSN of under 400nm in average diameter (DLS by methods described herein) after loadingof CRISPR-Cas9/gRNA cargo. See FIG. 18A. All particle types that weretested had an average diameter of 2 nm pores or greater. Hexagonalprisms had pore size of 2.5 nm, Stober particles are non-porous,Stellate particules were 12 nm and dendritic particles were either 8 nmor 18 nm in average pore size. These Examples were assayed by measuring% Green Fluorescent Protein (GFP) positive cells after 72-hour exposureof A549 reporter cells to 40, 60, and 80 μg/mL of the CRISPR-Cas9 loadedLC-MSN (FIG. 17A).

While several of the other particle types, including the dendritic 8 nmpore and 18 nm pore, resulted in a good sized stable LC-MSN, editingefficiency was surprisingly high (FIG. 17C) in the stellate core types.A stellate particle core with pore size range of 6-10.5 nm was selectedto move forward with studies on CRISPR-RNP delivery.

Examples 12: Comparison of CRISPR-Cas9 Loaded LC-MSN (Example 3) toCRISPR-Cas-9 and Free Lipid Composition from Example 2 with No CoreParticles

While the liposome composition in Example 2 mixed with CRISPR-Cas9 RNPresulted in a low amount of editing in the absence of the MSN, editingincreased substantially with the use of the stellate LC-MSNs of Example3. See FIG. 17B. The figure also includes a comparison with NiNTAfunctionalized particles. The NiNTA particles were MSN coresfunctionalized to capture Cas9 that is 6×HIS tagged. This is anothermethod used to load the particles with CRISPR RNPs.

All publications, patents, and patent applications mentioned in thisspecification are incorporated herein by reference to the same extent asif each independent publication or patent application was specificallyand individually indicated to be incorporated by reference.

What has been described above includes examples of one or moreembodiments. It is, of course, not possible to describe everyconceivable modification and alteration of the above devices ormethodologies for purposes of describing the aforementioned aspects, butone of ordinary skill in the art can recognize that many furthermodifications and permutations of various aspects are possible.Accordingly, the described aspects are intended to embrace all suchalterations, modifications, and variations that fall within the spiritand scope of the appended claims. Furthermore, to the extent that theterm “includes” is used in either the detailed description or theclaims, such term is intended to be inclusive in a manner similar to theterm “comprising” as “comprising” is interpreted when employed as atransitional word in a claim. The term “consisting essentially” as usedherein means the specified materials or steps and those that do notmaterially affect the basic and novel characteristics of the material ormethod. Unless the context indicates otherwise, all percentages andaverages are by weight. If not specified above, the properties mentionedherein may be determined by applicable NIST standards, or if an NISTstandard does not exist for the property, then NCL, and then ASTMstandards may be used, if none of the above standards are available, themost commonly used standard known by those of skill in the art may beused. The articles “a,” “an,” and “the,” should be interpreted to mean“one or more” unless the context indicates the contrary.

1. A construct comprising: a core comprising an external surface and aplurality of pores, wherein an average dimension of the plurality ofpores is greater than about 2 nm; a cargo disposed in a pore of theplurality of the pores, the cargo comprising one or more selected fromthe group consisting of: peptides, proteins, nucleic acids, mRNA,aptamers, antibodies, pharmaceuticals, antisense oligonucleotides,alpha/flavi virus inhibitors, coronavirus inhibitors, carbohydrates,dyes, and markers; and a coating coupled to the core, wherein thecoating comprises a cationic lipid, a pegylated lipid, a zwitterioniclipid, and a sterol; wherein the coating comprises a molar ratio ofabout 1 cationic lipid to 1 zwitterionic lipid to 0.9 sterol to 0.15PEGylated lipid, wherein each molar ratio optionally varies by aboutplus or minus 10%; or wherein the cationic lipid is1,2-dioleoyl-3-trimethylammonium-propane, the zwitterionic lipid is1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, the sterol ischolesterol, and the PEGylated lipid is1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy-(polyethyleneglycol)-2000].
 2. The construct of claim 1, wherein the coatingcomprises a molar ratio of about 1 cationic lipid to 1 zwitterioniclipid to 0.9 sterol to 0.15 PEGylated lipid, wherein each molar ratiooptionally varies by about plus or minus 10%.
 3. (canceled)
 4. Theconstruct of claim 1, further comprising a pharmaceutically acceptableexcipient.
 5. The construct of claim 1, wherein the core is a stellatemesoporous silica nanoparticle.
 6. The construct of claim 1, wherein theaverage diameter of the plurality of pores is of from about 3 nm toabout 20 nm as determined by porosimetry with nitrogenadsorption-desorption analysis.
 7. The construct of claim 6, wherein thecore has an average diameter of about 75 to about 400 nm.
 8. A method oftreating a subject, the method comprising: administering to a subject inneed thereof, an effective amount of a construct, the constructincluding: a core comprising an external surface and a plurality ofpores, wherein an average dimension of the plurality of pores is greaterthan about 2 nm; a cargo disposed in a pore of the plurality of pores,the cargo comprising a CRISPR Cas9 component, or a nucleic acid sequenceencoding a CRISPR Cas9 component; and a coating coupled to the core,wherein the coating comprises a cationic lipid, a PEGylated lipid, azwitterionic lipid, and a sterol.
 9. The method of claim 8, wherein thecore is a stellate mesoporous silica nanoparticle.
 10. The method ofclaim 8, wherein the average diameter of the plurality of pores is offrom about 3 nm to about 20 nm as determined by porosimetry withnitrogen adsorption-desorption analysis.
 11. The method of claim 8,wherein the core has an average diameter of about 75 to about 400 nm.12. The method of claim 9, wherein the mesoporous silica nanoparticle ismonodisperse in particle diameter, wherein about 90% of the distributionlies within about 5% of the median diameter, as measured by dynamiclight scattering.
 13. The method of claim 8, wherein the coatingcomprises about 10 to about 50 mol % of the cationic lipid andzwitterionic lipid, about 5 to about 45 mol. % of the sterol, and about2 to 8 mol. % of the PEGylated lipid.
 14. The method of claim 8, whereinthe coating comprises about 20 to about 40 mol % of the cationic lipidand zwitterionic lipid, about 10 to about 35 mol. % of the sterol, andabout 2.5 to 6 mol. % of the PEGylated lipid.
 15. The method of claim 8,wherein the coating comprises a molar ratio of about 1 cationic lipid to1 zwitterionic lipid to 0.9 sterol to 0.15 PEGylated lipid, wherein eachmolar ratio optionally varies by about plus or minus 10%.
 16. The methodof claim 8, wherein the cationic lipid is1,2-dioleoyl-3-trimethylammonium-propane, the zwitterionic lipid is1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, the sterol ischolesterol, and the PEGylated lipid is1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy-(polyethyleneglycol)-2000].
 17. The method of claim 8, wherein the sterol is selectedfrom the group consisting of: cholesterol, desmosterol, diplopterol,cholestanol, cholic acid, 12-deoxycholic acid, 7-deoxycholic acid, or aderivative thereof, and mixtures thereof and conjugated forms thereof.18. The method of claim 8, wherein the coating comprises a molar ratioof 1 cationic lipid to 1 zwitterionic lipid to 0.9 sterol to 0.15PEGylated lipid, wherein each molar ratio optionally varies by aboutplus or minus 10%; and wherein the cationic lipid is1,2-dioleoyl-3-trimethylammonium-propane, the zwitterionic lipid is1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, the sterol ischolesterol, and the PEGylated lipid is1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy-(polyethyleneglycol)-2000].
 19. The method of claim 8, wherein the cargo of theconstruct is configured to bind to a target sequence of the subject. 20.The method of claim 8, wherein the cargo is a coronavirus inhibitor. 21.The construct of claim 1, wherein the cargo is a coronavirus inhibitor.